Muc1 and galectin-3

ABSTRACT

The invention provides methods of identifying and making compounds that inhibit the interaction between MUC1 and galectin-3. Also embraced by the invention are in vivo and in vitro methods of inhibiting such an interaction and of inhibiting the expression of galectin-3 by a cell.

This application is continuation of U.S. patent application Ser. No.12/517,762 filed as a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2007/086760, filed Dec. 7, 2007,which claims the benefit of priority to U.S. provisional patentapplication Ser. No. 60/873,847, filed Dec. 8, 2006. The entire contentof each of the above referenced disclosures is specifically incorporatedherein by reference.

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an SCII compliant text file named “GENUP0019USC1ST25”, created onApr. 16, 2013 and having a size of ˜9 kilobytes. The content of theaforementioned file is hereby incorporated by reference in its entirety.

This invention was made with Government support under grant CA97098awarded by National Cancer Institute of the National Institutes ofHealth. The Government has certain rights in the invention.

1. FIELD OF THE INVENTION

This invention relates to regulation of cell growth, and moreparticularly to the regulation of cancer cell growth.

2. DESCRIPTION OF RELATED ART

The MUC1 protein is overexpressed by greater than 800,000 of the 1.4million tumors diagnosed in the United States each year.

Galectins are a family of lectins that contain conservedcarbohydrate-recognition domains (CRDs) of about 130 amino acids withspecificity for β-galactosides found on both N- and O-linked glycans(Liu and Rabinovich, 2005). Of the 15 known mammalian galectins, thewidely expressed galectin-3 is a structurally unique member with anN-terminal domain (ND) of repetitive sequences rich in proline, glycineand tyrosine residues upstream to the CRD. The ND lacks acarbohydrate-binding function, but is essential for the biologicactivity of galectin-3 (Seetharaman et al., 1998).

Galectin-3 overexpression has been found to be associated with thedevelopment of human malignancies (Liu and Rabinovich (2005; van denBrule et al. (2004); Califice et al. (2004). Cytosolic galectin-3 istargeted to the plasma membrane and released into the extracellularspace where it functions in the regulation of cell migration andadhesion (Ochieng et al., 2004). Galectin-3 is also targeted to thenucleus, where it is involved in regulating pre-mRNA splicing (Pattersonet al., 2004) and activation of certain transcription factors (Lin etal., 2002; Walzel et al., 2002). As found with MUC1 (Huang et al.,2005), galectin-3 binds directly to β-catenin and induces thetranscriptional activity of Tcf-4 (Shimura et al., 2004).

Like MUC1 (Ren et al., 2004; Wei et al., 2005), galectin-3 blocks theapoptotic response of breast cancer cells to genotoxic anti-canceragents (Takenaka et al., 2004a). The anti-apoptotic activity ofgalectin-3 is regulated by casein kinase 1-mediated phosphorylation ofnuclear galectin-3 on Ser-6 (Yoshii et al., 2002) and thereby its exportto the cytoplasm (Takenaka et al., 2004b). Translocation of galectin-3to mitochondria is associated with inhibition of cytochrome c releasewhich contributes to protection against the induction of apoptosis(Matarrese et al., 2000; Yu et al., 2002). Galectin-3 also contains anAsn-Trp-Gly-Arg (NWGR) motif that is conserved in the BH1 domain of theBc1-2 family members. This motif is of importance to the galectin-3anti-apoptotic function (Akahani et al., 1997). Little is known aboutthe regulation of galectin-3 expression in cancer cells.

SUMMARY OF THE INVENTION

The inventors have found that MUC1-C is N-glycosylated, binds togalectin-3, and induces galectin-3 expression by a post-transcriptionalmechanism. This application demonstrates that MUC1 and galectin-3function as part of a regulatory loop in which (i) N-glycosylation ofMUC1-C is necessary for suppression of miR-322 and thereby upregulationof galectin-3, and (ii) binding of galectin-3 to N-glycosylated MUC1 isof functional importance to integration of MUC1 with the epidermalgrowth factor receptor (EGFR) signaling pathway.

The invention includes methods for identifying compounds useful forinhibiting the interaction between MUC1-C and galectin-3. Such compoundscan be useful for directly promoting apoptosis of MUC1-expressing cancercells, for enhancing the efficacy of genotoxic chemotherapeutic agentsagainst such cancer cells, and as anti-cancer prophylactic agents. Alsoincluded in the invention are methods of inhibiting the interactionbetween galectin-3 and MUC1 in which cells (e.g., carcinoma cells suchas breast carcinoma cells) are contacted with compounds that inhibit theinteraction between MUC1 and galectin-3. While the experiments describedherein were generally performed with human MUC1, galectin-3, and cells,it is understood that the methods described herein can be performed withcorresponding molecules from any of the mammalian species recited below.

More specifically, the invention provides methods of identifyingcompounds that inhibit binding of MUC1-C to galectin-3. The methodsinclude: (a) providing a MUC1-C test agent; (b) providing a galectin-3test agent that binds to the MUC1-C test agent; (c) contacting theMUC1-C test agent with the galectin-3 test agent in the presence of atest compound under conditions that permit the binding of the MUC1-Ctest agent with the galectin-3 test agent in absence of the testcompound; and (d) determining whether the test compound inhibits bindingof the MUC1-C test agent to the galectin-3 test agent. The contactingcan be carried out in a cell-free system, in a cell, or on the surfaceof a cell. In some embodiments, the MUC1-C test agent comprises apeptide fragment of MUC1-C (e.g., all or part of the extracellulardomain of MUC1-C N-glycosylated at residue N36). In some embodiments,the MUC1-C test agent is a polypeptide (e.g., a soluble polypeptide)comprising a peptide fragment of the extracellular domain of MUC1-Cfused to an immunoglobulin Fc region.

Also featured are methods of identifying compounds that inhibitactivation of galectin-3 expression by MUC1. The methods include (a)providing a cell that expresses MUC1; (b) contacting the cell with atest compound under conditions that permit the expression of galectin-3in absence of the test compound; and (d) determining whether the testcompound inhibits expression of galectin-3. In some embodiments, thecell comprises a nucleic acid sequence comprising a 3′ untranslatedregion of galectin-3 operably linked to a sequence encoding a reporterpolypeptide (e.g., an enzyme or fluorescent polypeptide). In someembodiments, the test compound is microRNA-322 (miR-322).

Also featured are methods of generating compounds that inhibit theinteraction between MUC1-C and galectin-3. The methods include: (a)providing the three-dimensional structure of a molecule comprising apeptide fragment of galectin-3 or a peptide fragment of MUC1-C; (b)designing, based on the three dimensional structure, a compoundcomprising a region that inhibits the interaction between MUC1-C andgalectin-3; and (c) producing the compound. The methods can furtherinclude determining whether the compound generated inhibits theinteraction between MUC1-C and galectin-3.

Further, the invention includes processes of manufacturing compounds byperforming any of the above methods and, after determining that acompound inhibits the interaction between MUC1-C and galectin-3 orinhibits the activation of galectin-3 expression by MUC1, manufacturingthe compound.

Still further, the invention includes compounds (e.g., small molecules,polypeptides, and nucleic acids) identified, generated, or manufacturedby the above methods.

In another aspect, the invention features methods of inhibiting bindingof MUC1-C to galectin-3 or inhibiting association of MUC1 and EGFR in acancer cell that expresses MUC1-C by contacting the cancer cell with acompound (e.g., a small molecule, polypeptide, or polynucleotide) thatinhibits binding of galectin-3 to the extracellular domain of MUC1-Cand/or inhibits activation of galectin-3 expression by MUC1. In someembodiments, the compound includes: a peptide fragment of MUC1-C orgalectin-3; a peptide fragment of the extracellular domain of MUC1-C; apeptide fragment of the extracellular domain of MUC1-C fused to animmunoglobulin Fc region; all or part of the carbohydrate binding domainof galectin-3; or all or part of amino acids 63-250 or 117-244 ofgalectin-3 (SEQ ID NO:2). In some embodiments, the compound is anantibody, or an antibody fragment, that binds to galectin-3 or theextracellular domain of MUC1-C (e.g., the extracellular domain of MUC1-CN-glycosylated at residue N36). In further embodiments, the compound isa β-galactoside carbohydrate (e.g., lactose) or a carbohydrate analogthat binds to galectin-3. In further embodiments, the compound is aninhibitor of MUC1-C glycosylation or a compound that reduces theglycosylation of MUC1-C.

In other aspects, the invention includes methods of inhibitingexpression of galectin-3 in cancer cells that express MUC1. The methodsinclude (a) identifying a subject as having a cancer comprising a cancercell that expresses MUC1; and (b) introducing into the cell a compoundthat inhibits the expression of MUC1. In some embodiments, the compoundis a nucleic acid. Exemplary nucleic acids include antisense nucleicacids, small interfering RNAs (siRNAs), and nucleic acids that directthe expression of an antisense nucleic acid or siRNA. In someembodiments, the compound is a nucleic acid that includes a nucleic acidhaving the sequence of miR-322. In some embodiments, the step ofintroducing includes administration of the nucleic acid to the cancercell and uptake of the nucleic acid by the cancer cell. In someembodiments, the step of introducing includes administering to amammalian subject (e.g., a human subject), and uptake by the cancer cellof, a nucleic acid: (i) from which sense and anti-sense strands of thesiRNA can be transcribed under the direction of separate TREs; or (ii)from which both sense and anti-sense strands of the siRNA can betranscribed under the direction of a single TRE.

The cancer cell can be in a mammalian subject (e.g., a human subject).When the cancer cell is in a subject, the contacting can includedelivering (e.g., administering) the compound to the subject (e.g.,locally or systemically). When the compound is a protein (e.g., apolypeptide or an antibody), the administration can include directadministration or administering to the subject (a) a nucleic acidcomprising a nucleotide sequence encoding the protein, the nucleotidesequence being operably linked to a transcriptional regulatory element(TRE) (e.g., a DF3 enhancer) and/or (b) a recombinant cell (e.g., atransfected cell or a progeny of a transfected cell, made bytransfecting a cell derived from the subject) that is transfected withthe nucleic acid and that secretes the protein. When the compound is apolynucleotide (e.g., a nucleic acid aptamer, an antisense nucleic acid,a small interfering RNA, or a microRNA), the administration can includedirect administration or administering to the subject a nucleic acidcomprising a nucleotide sequence encoding the polynucleotide operablylinked to a transcriptional regulatory element (TRE) (e.g., a DF3enhancer).

Exemplary cancer cells that can be the target of the methods describedherein include cells of a cancer selected from the group consisting ofbreast cancer, lung cancer, colon cancer, pancreatic cancer, renalcancer, stomach cancer, liver cancer, bone cancer, hematological cancer,neural tissue cancer, melanoma, ovarian cancer, testicular cancer,prostate cancer, cervical cancer, vaginal cancer, and bladder cancer.

Further, the invention includes methods of killing cancer cells bybefore, after, or at the same time as, performing the above methods,exposing the cells to one or more genotoxic agents, e.g., one or moreforms of ionizing radiation or one or more chemotherapeutic agents(e.g., cisplatin, carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide, verapamil, podophyllotoxin,tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin,methotrexate, or an analog of any of the aforementioned).

In other aspects, the invention includes methods of inhibitingexpression of galectin-3 in cancer cells that expresses MUC1. Themethods include (a) identifying a subject as having a cancer comprisinga cancer cell that expresses MUC1; and (b) introducing into the cell anucleic acid that inhibits the expression of MUC1. Exemplary nucleicacids include antisense nucleic acids, small interfering RNAs (siRNAs),and nucleic acids that direct the expression of an antisense nucleicacid or siRNA. In some embodiments, the step of introducing includesadministration of the nucleic acid to the cancer cell and uptake of thenucleic acid by the cancer cell. In some embodiments, the step ofintroducing includes administering to a mammalian subject (e.g., a humansubject), and uptake by the cancer cell of, a nucleic acid: (i) fromwhich sense and anti-sense strands of the siRNA can be transcribed underthe direction of separate TREs; or (ii) from which both sense andanti-sense strands of the siRNA can be transcribed under the directionof a single TRE.

In further aspects, the invention features methods of promotingapoptosis of a cell (e.g., a cancer cell) that include determiningwhether the cell expresses MUC1; and if the cell expresses MUC1,contacting the cell with a compound that inhibits phosphorylation ofgalectin-3 by casein kinase 1.

Other methods featured by the invention are methods of diagnosing a testcell that include measuring the level of galectin-3 or a MUC1-galectin-3complex in a test cell, wherein an enhanced level of galectin-3 orMUC1-galectin-3 complex in the test cell is an indication that the testcell is a cancer cell.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. The MUC1-C and galectin-3 molecules andtest agents used in any of the methods of the invention can contain orbe wild-type proteins or can be variants that have not more than fifty(e.g., not more than: one, two, three, four, five, six, seven, eight,nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acidsubstitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine. All that is required as that: (i) suchvariants of MUC1-C have at least 25% (e.g., at least: 30%; 40%; 50%;60%; 70%; 75%; 80%; 85%; 90%; 95%; 97%; 98%; 99%; 99.5%, or 100% or evengreater) of the ability of wild-type MUC1-C to bind to galectin-3; and(ii) such variants of a galectin-3 have at least 25% (e.g., at least:30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 97%; 98%; 99%; 99.5%,or 100% or even greater) of the ability of wild-type galectin-3 to bindto MUC1-C.

As used herein, a “MUC1-C test agent” contains, or is, (a) full-length,wild-type mature MUC1-C, (b) a part of MUC1-C that is shorter thanfull-length, wild-type, mature MUC1-C, or (c) (a) or (b) but with one ormore (see above) conservative substitutions. “Parts of a MUC1-C” includefragments (e.g., MUC1C-ter or the extracellular domain (ED) of MUC1-C)as well deletion variants (terminal as well internal deletions) ofMUC1-C. Deletion variants can lack one, two, three, four, five, six,seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacid segments (of two or more amino acids) or non-contiguous singleamino acids. MUC1-C test agents can include internal or terminal(carboxy or amino) irrelevant amino acid sequences (e.g., sequencesderived from other proteins or synthetic sequences not corresponding toany naturally occurring protein). These added irrelevant sequences willgenerally be about 1-50 (e.g., two, four, eight, ten, 15, 20, 25, 30,35, 40, or 45) amino acids in length. MUC1-C test agents other thanfull-length, wild-type, mature MUC1-C will have at least 50% (e.g., atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, at least 99%, at least 99.5%, or 100% or more)of the ability of the full-length, wild-type, mature MUC1-C to bind togalectin-3.

As used herein, a “galectin-3 test agent” contains, or is, (a)full-length, wild-type mature galectin-3, (b) a part of galectin-3 thatis shorter than full-length, wild-type, mature galectin-3, or (c) (a) or(b) but with one or more (see above) conservative substitutions. “Partsof a galectin-3” include fragments (e.g., amino acids 63-250 or thecarbohydrate binding domain of galectin-3) as well deletion variants(terminal as well internal deletions) of galectin-3. Deletion variantscan lack one, two, three, four, five, six, seven, eight, nine, ten, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two ormore amino acids) or non-contiguous single amino acids. Galectin-3 testagents can include internal or terminal (carboxy or amino) irrelevantamino acid sequences (e.g., sequences derived from other proteins orsynthetic sequences not corresponding to any naturally occurringprotein). These added irrelevant sequences will generally be about 1-50(e.g., two, four, eight, ten, 15, 20, 25, 30, 35, 40, or 45) amino acidsin length. Galectin-3 test agents other than full-length, wild-type,mature galectin-3 will have at least 50% (e.g., at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least98%, at least 99%, at least 99.5%, or 100% or more) of the ability ofthe full-length, wild-type, mature galectin-3 to bind to MUC1-C.

As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativeare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device and/ormethod being employed to determine the value.

As used herein the specification, “a” or “an” may mean one or more,unless clearly indicated otherwise. As used herein in the claim(s), whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A, 1B, 1C, 1D, 1E. MUC1 upregulates galectin-3 expression.1A—Lysates from the indicated cells were immunoblotted with anti-MUC1-C,anti-galectin-3 and anti-β-actin. 1B—The empty pIRES-puro vector andpIRES-puro-MUC1 were stably transfected into BT549 breast cancer cells.Lysates from the transfectants were immunoblotted with the indicatedantibodies. The BT549/MUC1-A and -B cells represent two separatelyisolated clones. 1C—Lysates from ZR-75-1/vector-A andZR-75-1/MUC1siRNA#2-A cells were immunoblotted with the indicatedantibodies. 1D—Lysates from DU145 prostate cancer cells stablyexpressing the pRNA-U6.1/Neo CsiRNA or MUC1siRNA#2 were immunoblottedwith the indicated antibodies. 1E—DU145 cells stably expressingMUC1siRNA#4 were transfected with pIRES-puro2 or pIRES-puro2-MUC1-C.Lysates were immunoblotted with the indicated antibodies. DU145/CsiRNAcells were included for comparison.

FIG. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21. MUC1 stabilizes galectin-3transcripts. 2A, 2B—BT549/vector and BT549/MUC1 cells (A) orZR-75-1/vector and ZR-75-1/MUC1siRNA (stably silenced for MUC1) cells(B) were analyzed for galectin-3 and GAPDH mRNA levels bysemi-quantitative RT-PCR (left) and quantitative RT-PCR (right). Thequantitative RT-PCR results (mean±SD from three replicates) areexpressed as the relative galectin-3 mRNA levels (normalized to GAPDH)compared to that obtained with BT549/vector (A) and ZR-75-1/MUC1siRNA(B) cells (assigned a value of 1). 2C—DU145/CsiRNA and DU145/MUC1siRNA#4cells were analyzed for galectin-3 and GAPDH mRNA levels by quantitativeRT-PCR (left). DU145/MUC1siRNA#4 cells were transfected with pIRES-puro2or pIRES-puro2-MUC1-C and then analyzed by quantitative RT-PCR (right).The results (mean±SD from three replicates) are expressed as therelative galectin-3 mRNA levels (normalized to GAPDH) compared to thatobtained with DU145/MUC1siRNA#4 (left) and DU145/MUC1siRNA#4 transfectedwith pIRES-puro2 (right) cells (assigned a value of 1). The asterisk (*)denotes a significant difference at p<0.05 as compared to control.2D—BT549/vector and BT549/MUC1 cells were treated with actinomycin D andthen harvested at the indicated times. RNA was analyzed for galectin-3and GAPDH mRNA levels by quantitative RT-PCR. The results are expressedas relative galectin-3 mRNA levels for BT549/vector (O) and BT549/MUC1 () cells. 2E—Construction of the galectin-3 3′UTR-luciferase reporterplasmid. BT549/vector and BT549/MUC1 cells (left) or ZR-75-1/vector andZR-75-1/MUC1siRNA cells (right) were transfected with thepMIR-Gal-3(3′UTR) and, as a control, pMIR-β-galactosidase plasmids. Thecells were analyzed for luciferase and β-gal activities at 48 h aftertransfection. The results (mean±SD of three experiments) are expressedas the relative luciferase activity (normalized to β-gal) compared tothat in BT549/vector (left) or ZR-75-1/MUC1siRNA (right) cells (assigneda value of 1). The asterisk (*) denotes a significant difference atp<0.05 as compared to control. 2F—The indicated BT549 cells weretransfected with pGal-3 (−3000/+141)—Luc and pcDNA-LacZ plasmids. 2G,2H—ZR-75-1/MUC1siRNA and ZR-75-1/vector cells were transfected with theindicated pGal-Luc constructs and pcDNA-LacZ. The cells were assayed forluciferase and β-gal activities at 24 h after transfection. The results(mean±SD from three experiments) are expressed as relative galectin-3promoter activity compared to that obtained with the BT549/vector (2F)or ZR-75-1/MUC1siRNA (2G and 2H) cells (assigned a value of 1).2I—Galectin-3 gene transcription was assayed for the indicated BT549(left) and ZR-75-1 (right) cells in run-on assays using the β-actin geneas an internal control. The results (mean±SD from three replicates) areexpressed as the relative change in the abundance of newly transcribedgalectin-3 mRNA.

FIG. 3A, 3B, 3C, 3D, 3E, 3F, 3G. MUC1 downregulates miR-322 and therebyincreases stability of galectin-3 mRNA. 3A—Model of the pre-miRstem-loop structure encoding miR-424 and miR-322 (SEQ ID NO:22).3B—Sequence alignment of human miR-322 with mouse and rat miR-322 andwith the galectin-3 3′UTR (SEQ ID NO:23 and SEQ ID NO:24). 3C—Northernblot analyses of RNA from the indicated BT549, ZR-75-1 and DU145 cellsprobed for miR-322 (upper panels) and U6 snRNA as a loading control(lower panels). 3D—BT549/vector cells were transfected with an antisense2′-O-methyl oligoribonucleotide targeted against miR-322 or a scrambled2′-β-methyl oligoribonucleotide as a control for 48 h. The cells weretreated with actinomycin D and harvested at the indicated times. TotalRNA was analyzed for galectin-3 and GAPDH mRNA levels by quantitativeRT-PCR. The results are expressed as relative galectin-3 mRNA levels forBT549 cells transfected with the scrambled oligo (O) or the anti-miR-322( ) 3E—BT549/MUC1 cells were transfected with a pre-mIR-322 or ascrambled miRNA and selected in the presence of blasticidin. Cells wereassayed for galectin-3 mRNA (left) and protein (right). The results(mean±SD for three replicates) are expressed as the relative galectin-3mRNA levels (normalized to GAPDH) compared to that obtained with thescrambled miRNA (assigned a value of 1)(left). The asterisk (*) denotesa significant difference at p<0.01 as compared to control (left).3F—BT549/vector cells were transfected with an antisense 2′-O-methyloligoribonucleotide targeted against miR-322 or a scrambled 2′-O-methyloligoribonucleotide as a control for 72 h. The cells were analyzed forgalectin-3 and GAPDH mRNA levels by quantitative RT-PCR (left) orgalectin-3 and β-actin protein levels by immunoglotting (right). TheRT-PCR results (mean±SD) from three replicates) are expressed as therelative galectin-3 mRNA levels (normalized to GAPDH) compared to thatobtained with the scrambled miRNA (assigned a value of 1). The asterisk(*) denotes a significant different at p<0.01 as compared to normal.3G—BT549/MUC1 cells were transfected with a pre-mIR-322 or a scrambledmiRNA and selected in the presence of blasticidin. Nuclei were assayedfor galectin-3 and β-actin gene transcription in run-on assays. Theresults (mean±SD from three replicates) are expressed as the relativechange in the abundance of newly transcribed galectin-3 mRNA.

FIG. 4A, 4B, 4C, 4D, 4E, 4F. Galectin-3 associates with the MUC1-Cextracellular domain. 4A—Lysates from ZR-75-1 cells wereimmunoprecipitated with anti-MUC1-C and, as a control, a non-specificIgG (left). The precipitates were immunoblotted with the indicatedantibodies. ZR-75-1 cell lysates were incubated with GST orGST-galectin-3 bound to glutathione beads (right). The adsorbates wereimmunblotted with anti-MUC1-C. Input of the GST and GST-galectin-3proteins was assessed by Coomassie blue staining. 4B—Schematicrepresentation of the transmembrane MUC1-C subunit and amino acidsequence of the extracellular domain (MUC1-C/ED) (SEQ ID NO:25).4C—Lett. ZR-75-1 cells were grown in 0.1% FBS for 2 days. Cell surfacebinding proteins were released in salt solution, diluted and passedthrough hFc or hFc-MUC1-C/ED columns. The adsorbed proteins were elutedand analyzed by SDS-PAGE and silver staining. The 26 kDa protein wassubjected to trypsin digestion. Analysis of the tryptic peptides byLC-MS demonstrated identity with galectin-3. Right. hFc andhFc-MUC1-C/ED were incubated with purified galectin-3 and precipitatedwith protein G-sepharose. The adsorbates were immunoblotted withanti-galectin-3 (upper panel). The input proteins were stained withCoomassie blue (lower panel). 4D—Schema of galectin-3 is shown with theregions expressed as GST fusion proteins. hFc-MUC1-C/ED was incubatedwith GST or the indicated GST-galectin-3 fusion proteins. Adsorbates toglutathione beads were immunoblotted with anti-MUC1-C/ED. Input proteinswere stained with Coomassie blue. 4E—hFc-MUC1-C/ED was injected atconcentrations ranging from 0 nM to 100 nM at a constant flow rate of 30μl/min over a galectin-3 immobilized chip (4E), over a galectin-3(63-250) immobilized chip (4F) and over a control dextran surface. Theassociation was monitored for 200 seconds and the dissociation observedfor 500 seconds. The dark curve represents the fit of data to 1:1binding with a drifting base model.

FIG. 5A, 5B, 5C, 5D, 5E, 5F. Glycosylation of MUC1-C on Asn-36 isnecessary for galectin-3 binding. 5A—Lysates from BT549/MUC1 (left) andZR-75-1 (right) cells were immunoprecipitated with anti-MUC1-C. Theprecipitates were left untreated or digested with N-glycosidases andthen immunoblotted with anti-MUC1-C. 5B—MUC1-C was transiently expressedin wild-type CHO-K1 cells or the glycosylation-deficient Led 1 and Lec8variants. Lysates were immunoblotted with anti-MUC1-C (left). Thelysates were also incubated with GST-galectin-3 and the precipitatesimmunoblotted with anti-MUC1-C (right). 5C—Lett. Lysates from BT549cells stably expressing MUC1-C or MUC1-C(N36A) were immunoblotted withanti-MUC1-C. Right. Lysates from BT549/MUC1-C cells wereimmunoprecipitated with anti-MUC1-C. The precipitates were leftuntreated or digested with N-glycosidases and immunoblotted withanti-MUC1-C. 5D—Lysates from BT549/MUC1-C and BT549/MUC1-C(N36A) cellswere incubated with GST-galectin-3 and the adsorbates were immunoblottedwith anti-MUC1-C. 5E—ZR-75-1 cells were incubated with sucrose orlactose in complete medium for 24 h and then with sucrose and lactosefor an additional 24 h in 0.1% serum. The cells were then stimulatedwith EGF for 5 min. 5F—ZR-75-1 cells were transfected with a controlsiRNA or a galectin-3 siRNA pool for 48 h and then grown in the presenceof 0.1% serum for 24 h. The cells were then stimulated with EGF for 5min. Cells were analyzed by confocal microscopy after fixation andstaining with anti-EGFR and anti-MUC1-N.

FIG. 6A, 6B, 6C, 6D. Glycosylation of MUC1-C on Asn-36 is associatedwith upregulation of galectin-3 expression. 6A—Lysates from BT549 cellsstably expressing the empty vector, MUC1-C or MUC1-C(N36A) wereimmunoblotted with the indicated antibodies. 6B—The indicated BT549cells were analyzed for galectin-3 and GAPDH mRNA levels bysemi-quantitative RT-PCR (left) and real time RT-PCR (right). The realtime RT-PCR results (mean±SD from three replicates) are expressed as therelative galectin-3 mRNA levels (normalized to GAPDH) compared to thatobtained with BT549/vector cells (assigned a value of 1). 6C—Theindicated BT549 cells were transfected with pMIR-Gal-3(3′UTR) andpMIR-β-gal plasmids. The cells were assayed for luciferase and β-galactivities at 48 h after transfection. The results (mean±SD of threeexperiments) are expressed as the relative luciferase activity(normalized to β-gal) compared to that in BT549/vector cells.6D—Northern blot analysis of RNA from the indicated BT549 cells probedfor miR-322 and U6 snRNA.

FIG. 7A, 7B, 7C, 7D. Interaction between MUC1 and EGFR is mediated bygalectin-3. 7A—ZR-75-1 cells were incubated with sucrose or lactose incomplete medium for 24 h and then with sucrose and lactose for anadditional 24 h in 0.1% serum. The cells were then stimulated with EGFfor 5 min. 7B—ZR-75-1 cells were transfected with a control siRNA or agalectin-3 siRNA pool for 48 h and then grown in the presence of 0.1%serum for 24 h. The cells were then stimulated with EGF for 5 min.Anti-MUC1-N precipitates were immunoblotted with the indicatedantibodies (A and B). Lysates were also directly immunoblotted withanti-galectin-3 (B). 7C, 7D—ZR-75-1 cells were transfected with apre-mIR-322 or a scrambled miRNA and selected in blasticidin. Lysateswere immunoblotted with the indicated antibodies (C). Anti-MUC1-Nprecipitates were immunoblotted with the indicated antibodies (D).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The MUC1 mucin-type, transmembrane glycoprotein is expressed on theapical borders of normal secretory epithelial cells (Kufe et al., 1984).With transformation and loss of polarity, MUC1 is aberrantlyoverexpressed on the entire cell surface in carcinomas of the breast,prostate, lung and other epithelia (Kufe et al., 1984). The MUC 1polypeptide undergoes autoproteolysis in the endoplasmic reticulum withthe generation of two subunits that in turn form a stable heterodimer(Ligtenberg et al., 1992); Levitin et al., 2005; Macao et al., 2006).The MUC1 N-terminal subunit (MUC1-N) consists in large part of variablenumbers of 20 amino acid tandem repeats that are subject to extensiveβ-glycosylation (Gendler et al., 1988; Siddiqui et al., 1988). MUC1-N istethered to the cell surface through noncovalent binding to thetransmembrane MUC1 C-terminal subunit, which consists of a 58 amino acidextracellular domain, a 28 amino acid transmembrane domain and a 72amino acid cytoplasmic tail (Merlo et al., 1989). MUC1-N extends beyondthe cell glycocalyx as part of a physical barrier that protectsepithelial cells from damage induced by free radicals, low pH, toxinsand other forms of stress that occur at the interface with the externalenvironment. MUC1-N can also be shed into this protective barrier,leaving MUC1-C at the cell surface as a putative receptor for signalingthe presence of stress to the interior of the cell. In this context,overexpression of MUC1 in transformed cells is associated withaccumulation of MUC1-C in the cytosol and targeting of this subunit tothe nucleus (Li et al., 2003; Li et al., 2003; Li et al., 2003; Wen etal., 2003) and mitochondria (Ren et al., 2004; Ren et al., 2006). Insupport of a role for MUC1-C in signal transduction, the cytoplasmicdomain functions as a substrate for the epidermal growth factor receptor(Li et al., 2001), c-Src (Li et al., 2001) and glycogen synthase 3β (Liet al., 1998). Moreover, the MUC1-C cytoplasmic domain interactsdirectly with the Wnt pathway effector, β-catenin (Li et al., 1998;Yamamoto et al., 1997; Huang et al., 2005) and with the p53 tumorsuppressor (Wei et al., 2005). Studies have demonstrated thatoverexpression of MUC1 is sufficient to confer resistance tostress-induced apoptosis (Li et al., 2003; Ren et al., 2004; Wei et al.,2005; Raina et al., 2004), anchorage-independent growth andtumorigenicity (Li et al., 2003; Huang et al., 2005; Schroeder et al.,2004). MUC1 associates with EGFR constitutively, and this interaction isstimulated by EGF treatment (Li et al., 2001). The human MUC1-Cpolypeptide sequence is:

(SEQ ID NO: 1) SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVS AGNGGSSLSYTNPAVAATSANL

Galectins are a family of lectin proteins that contain conservedcarbohydrate-recognition domains (CRDs) with specificity forβ-galactosides found on both N- and O-linked glycans (Liu andRabinovich, 2005). Of the fifteen known mammalian galectins, the widelyexpressed galectin-3 is a structurally unique member with an N-terminaldomain of 12 amino acids and repetitive sequences rich in proline,glycine and tyrosine upstream to the CRD. Cytosolic galectin-3 istargeted to the plasma membrane and released into the extracellularspace (Hughes (1999) Biochim. Biophys. Acta. 1473:172-185), where itfunctions in the regulation of cell migration and adhesion (Ochieng(2004) Glycoconj. J. 19:527-535). Galectin-3 is also targeted to thenucleus where it plays a role in regulating pre-mRNA splicing (Pattersonet al. (2004) Glycoconj J. 19:499-506) and contributes to the activationof diverse transcription factors (Lin et al., 2002; Dudas et al., 2002;Walzel et al., 2002; Paron et al., 2003). As with MUC1 (Huang et al.,2003), galectin-3 binds directly to β-catenin and induces thetranscriptional activity of Tcf-4 (Shimura et al., 2004). In addition,like MUC1 (Ren et al., 2004; Wei et al., 2005), galectin-3 blocks theapoptotic response of breast cancer cells to genotoxic anti-canceragents (Takenaka et al., 2004). The anti-apoptotic activity ofgalectin-3 is regulated by casein kinase 1-mediated phosphorylation ofnuclear galectin-3 on Ser-6 (Yoshii et al., 2002) and thereby its exportto the cytoplasm (Takenaka et al., 2004). Translocation of galectin-3 tomitochondria with inhibition of cytochrome c release contributes in partto protection against the induction of apoptosis (Matarrese et al.,2000; Yu et al., 2002). Galectin-3 also contains an Asp-Trp-Gly-Arg(NWGR; SEQ ID NO:8) motif that is conserved in the BH1 domain of theBcl-2 family members and is of importance to the galectin-3anti-apoptotic function (Akahani et al., 1997). Other studies havedemonstrated that galectin-3 induces transformation (Takenaka et al.,2003; Nangia-Makker et al., 1995) and is upregulated in diversecarcinomas (Takenaka et al., 2004). Studies have shown that EGFR iscross-linked at the cell surface by galectin-3 (Partridge et al., 2004).

The human galectin-3 polypeptide sequence is:

(SEQ ID NO: 2) MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYHGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSAPGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHR VKKLNEISKLGISGDIDLTSASYTMI

An exemplary carbohydrate-binding domain of galectin-3 includes aminoacid residues 117-244 of SEQ ID NO:2.

miRNAs are noncoding RNAs of about 22 nucleotides thatpost-transcriptionally regulate gene expression. Base-pairing of themiRNA with complementary sequences in a mRNA 3′UTR leads to suppressionof translation or decreases in mRNA stability (Bartel (2004).Translational suppression is a more commonly described mechanism ofmiRNA action; however, there are now an increasing number of reports ofmiRNA-mediated mRNA degradation (Bagga et al., 2005; Krutzfeldt et al.,2005; Lim et al., 2005; Wu and Belasco, 2005). Binding of the miRNA isassociated with accelerated deadenylation of the mRNA (Wu et al., 2006).Herein, miR-322 is identified as a putative regulator of galectin-3expression that is expressed in human cells. The precursor stem loopsequence for miR-322 is located at chromosome Xq26.3. The genomicannotation specifies overlapping of the miR-322 precursor sequence withthe 5′UTR of a hypothetical gene, designated MGC16121, indicating thatboth the pre-miRNA and the MGC16121 mRNA may originate from the sametranscript. In this regard, like miR-322, MUC1 suppresses MGC16121 mRNAlevels. Upstream to the MGC16121 5′UTR is a putative promoter with aTATA box and, within 2000 bp, potential binding sites for 39 differenttranscription factors. The sequence of human miR-322 as deduced from thesequence of the human genome is 5′-AAACGUGAGGCGCUGCUAUA-3′ (SEQ IDNO:4).

Methods of Screening for Inhibitory Compounds

The invention provides in vitro methods for identifying compounds (e.g.,small molecules or macromolecules) that inhibit binding of galectin-3 toMUC1-C.

These methods can be performed using: (a) isolated MUC1-C test agentsand galectin-3 test agents; (b) cells expressing both a MUC1-C testagent and a galectin-3 test agent; (c) cells expressing a MUC1-C testagent and different cells expressing a galectin-3 test agent; or (d)isolated MUC1-C test agents or galectin-3 test agents and cellsexpressing a galectin-3 test agent or a MUC1-C test agent.

The term “isolated” as applied to any of the above-listed polypeptidetest agents refers to a polypeptide, or a peptide fragment thereof,which either has no naturally-occurring counterpart or has beenseparated or purified from components which naturally accompany it,e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle,joint tissue, neural tissue, gastrointestinal tissue or tumor tissue(e.g., breast cancer or colon cancer tissue), or body fluids such asblood, serum, or urine. Typically, the polypeptide or peptide fragmentis considered “isolated” when it is at least 70%, by dry weight, freefrom the proteins and other naturally-occurring organic molecules withwhich it is naturally associated. Preferably, a preparation of a testagent is at least 80%, more preferably at least 90%, and most preferablyat least 99%, by dry weight, the test agent. Since a polypeptide that ischemically synthesized is, by its nature, separated from the componentsthat naturally accompany it, a synthetic polypeptide test agent is“isolated.”

An isolated polypeptide test agent can be obtained, for example, byextraction from a natural source (e.g., from tissues); by expression ofa recombinant nucleic acid encoding the polypeptide; or by chemicalsynthesis. A polypeptide test agent that is produced in a cellularsystem different from the source from which it naturally originates is“isolated,” because it will necessarily be free of components whichnaturally accompany it. The degree of isolation or purity can bemeasured by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

Prior to testing, any of the test agents can undergo modification, e.g.,phosphorylation or glycosylation, by methods known in the art. The testagents can be modified prior to isolation.

In methods of screening for compounds that inhibit binding of anisolated MUC1-C test agent to an isolated galectin-3 test agent, aMUC1-C test agent is contacted with a galectin-3 test agent in thepresence of one or more concentrations of a test compound and bindingbetween the two test agents in the presence and absence of the testcompound is detected and/or measured. In such assays neither of the testagents need be detectably labeled. For example, by exploiting thephenomenon of surface plasmon resonance, the MUC1-C test agent can bebound to a suitable solid substrate and the galectin-3 test agentexposed to the substrate-bound MUC1-C test agent in the presence andabsence of the compound of interest. Binding of the galectin-3 testagent to the MUC1-C test agent on the solid substrate results in achange in the intensity of surface plasmon resonance that can bedetected qualitatively or quantitatively by an appropriate instrument,e.g., a Biacore™ apparatus (Biacore International AB, Rapsgatan,Sweden). It will be appreciated that the experiment can be performed inreverse, i.e., with the galectin-3 test agent bound to the solidsubstrate and the MUC1-C test agent added to it in the presence of thetest compound.

Moreover, assays to test for inhibition of binding to MUC1-C can involvethe use, for example, of: (a) a single MUC1-C-specific “detection”antibody that is detectably labeled; (b) an unlabeled MUC1-C-specificantibody and a detectably labeled secondary antibody; or (c) abiotinylated MUC1-C-specific antibody and detectably labeled avidin. Inaddition, combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays. In these assays, the MUC1-binder test agent canbe immobilized on a solid substrate such as a nylon or nitrocellulosemembrane by, for example, “spotting” an aliquot of a sample containingthe test agent onto a membrane or by blotting onto a membrane anelectrophoretic gel on which the sample or an aliquot of the sample hasbeen subjected to electrophoretic separation. Alternatively, thegalectin-3 test agent can be bound to a substrate (e.g., the plasticbottom of an ELISA (enzyme-linked immunosorbent assay) plate well) usingmethods known in the art. The substrate-bound test agent is then exposedto the MUC1-C test agent in the presence and absence of the testcompound. After incubating the resulting mixture for a period of timeand at temperature optimized for the system of interest, the presenceand/or amount of MUC1-C test agent bound to the galectin-3 test agent onthe solid substrate is then assayed using a detection antibody thatbinds to the MUC1-C test agent and, where required, appropriatedetectably labeled secondary antibodies or avidin. It will beappreciated that instead of binding the galectin-3 test agent to thesolid substrate, the MUC1-C test agent can be bound to it. In this casebinding of the galectin-3 test agent to the substrate-bound MUC1-C istested by obvious adaptations of the method described above forsubstrate-bound MUC1-binder test agent.

The invention also features “sandwich” assays. In these sandwich assays,instead of immobilizing test agents on solid substrates by the methodsdescribed above, an appropriate test agent can be immobilized on thesolid substrate by, prior to exposing the solid substrate to the testagent, conjugating a “capture” test agent-specific antibody (polyclonalor mAb) to the solid substrate by any of a variety of methods known inthe art. The test agent is then bound to the solid substrate by virtueof its binding to the capture antibody conjugated to the solidsubstrate. The procedure is carried out in essentially the same mannerdescribed above for methods in which the appropriate test agent is boundto the solid substrate by techniques not involving the use of a captureantibody. It is understood that in these sandwich assays, the captureantibody should not bind to the same epitope (or range of epitopes inthe case of a polyclonal antibody) as the detection antibody. Thus, if amAb is used as a capture antibody, the detection antibody can be either:(a) another mAb that binds to an epitope that is either completelyphysically separated from or only partially overlaps with the epitope towhich the capture mAb binds; or (b) a polyclonal antibody that binds toepitopes other than or in addition to that to which the capture mAbbinds. On the other hand, if a polyclonal antibody is used as a captureantibody, the detection antibody can be either (a) a mAb that binds toan epitope that is either completely physically separated from orpartially overlaps with any of the epitopes to which the capturepolyclonal antibody binds; or (b) a polyclonal antibody that binds toepitopes other than or in addition to that to which the capturepolyclonal antibody binds. Assays which involve the use of a capture anda detection antibody include sandwich ELISA assays, sandwich Westernblotting assays, and sandwich immunomagnetic detection assays.

Suitable solid substrates to which the capture antibody can be boundinclude, without limitation, the plastic bottoms and sides of wells ofmicrotiter plates, membranes such as nylon or nitrocellulose membranes,polymeric (e.g., without limitation, agarose, cellulose, orpolyacrylamide) beads or particles.

Methods of detecting and/or for quantifying a detectable label depend onthe nature of the label and are known in the art. Appropriate labelsinclude, without limitation, radionuclides(e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H,³²P, or ¹⁴C), fluorescent moieties (e.g., fluorescein, rhodamine, orphycoerythrin), luminescent moieties (e.g., Qdot™ nanoparticles suppliedby the Quantum Dot Corporation, Palo Alto, Calif.), compounds thatabsorb light of a defined wavelength, or enzymes (e.g., alkalinephosphatase or horseradish peroxidase). The products of reactionscatalyzed by appropriate enzymes can be, without limitation,fluorescent, luminescent, or radioactive or they may absorb visible orultraviolet light. Examples of detectors include, without limitation,x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers.

Candidate compounds can also be tested for their ability to inhibitbinding of MUC1-C to galectin-3 in cells. The cells can either naturallyexpress an appropriate MUC1-C test agent and/or galectin-3 test agent ofinterest or they can recombinantly express either or both test agents.The cells can be normal or malignant and of any histological type, e.g.,without limitation, epithelial cells, fibroblasts, lymphoid cells,macrophages/monocytes, granulocytes, keratinocytes, or muscle cells.Suitable cell lines include those recited in the examples, e.g., breastcancer or colon cancer cell lines. The test compound can be added to thesolution (e.g., culture medium) containing the cells or, where thecompound is a protein, the cells can recombinantly express it. The cellscan optionally also be exposed to a stimulus of interest (e.g., a growthfactor such as EGF) prior to or after exposure of the cells to thecompound. Following incubation of cells expressing the test agents ofinterest in the absence or presence (optionally at variousconcentrations), physical association between the test agents can bedetermined microscopically using appropriately labeled antibodiesspecific for both test agents, e.g., by confocal microscopy.Alternatively, the cells can be lysed under non-dissociating conditionsand the lysates tested for the presence of physically associated testagents. Such methods include adaptations of those described usingisolated test agents. For example, an antibody specific for one of thetwo test agents (test agent 1) can be bound to a solid substrate (e.g.,the bottom and sides of the well of a microtiter plate or a nylonmembrane). After washing away unbound antibody, the solid substrate withbound antibody is contacted with the cell lysate. Any test agent 1 inthe lysate, bound or not bound to the second test agent (test agent 2),will bind to the antibody specific for test agent 1 on the solidsubstrate. After washing away unbound lysate components, the presence oftest agent 2 (bound via test agent 1 and the antibody specific for testagent 1 to the solid substrate) is tested for using a detectably labeledantibody (see above) specific for test agent 2. Alternatively, testagent 1 can be immunoprecipitated with an antibody specific for testagent 1 and the immunoprecipitated material can be subjected toelectrophoretic separation (e.g., by polyacrylamide gel electrophoresisperformed under non-dissociating conditions). The electrophoretic gelcan then be blotted onto a membrane (e.g., a nylon or a nitrocellulosemembrane) and any test agent 2 on the membrane detected and/or measuredwith a detectably labeled antibody (see above) specific for test agent 2by any of the above-described methods. It is understood that in theabove-described assays, test agent 1 can be either the MUC1-C test agentor the galectin-3 test agent or vice versa.

Methods of Designing and Producing Inhibitory Compounds

The invention also relates to using MUC1-C test agents and/or galectin-3test agents to predict or design compounds that can interact with MUC1-Cand/or galectin-3 and potentially thereby inhibit the ability of MUC1-Cto interact with an appropriate tumor progressor. One of skill in theart would know how to use standard molecular modeling or othertechniques to identify small molecules that would bind to “appropriatesites” on MUC1-C and/or tumor progressors. One such example is providedin Broughton (1997). Generally, an “appropriate site” on MUC1-C orgalectin-3 is a site directly involved in the physical interactionbetween the two molecule types. However, an “appropriate site” can alsobe an allosteric site, i.e., a region of the molecule not directlyinvolved in a physical interaction with another molecule (and possiblyeven remote from such a “physical interaction” site) but to whichbinding of a compound results (e.g., by the induction in aconformational change in the molecule) in inhibition of the binding ofthe molecule to another molecule

By “molecular modeling” is meant quantitative and/or qualitativeanalysis of the structure and function of protein-protein physicalinteraction based on three-dimensional structural information andprotein-protein interaction models. This includes conventionalnumeric-based molecular dynamic and energy minimization models,interactive computer graphic models, modified molecular mechanicsmodels, distance geometry and other structure-based constraint models.Molecular modeling typically is performed using a computer and may befurther optimized using known methods.

Methods of designing compounds that bind specifically (e.g., with highaffinity) to a region of MUC1-C that interacts with galectin-3 (e.g.,all or a part of the extracellular domain of MUC1-C) or a region ofgalectin-3 that binds to MUC1 (e.g., a region within the carbohydratebinding domain and/or all or a part of amino acid residues 63-250)typically are also computer-based, and involve the use of a computerhaving a program capable of generating an atomic model. Computerprograms that use X-ray crystallography data are particularly useful fordesigning such compounds. Programs such as RasMol, for example, can beused to generate a three dimensional model of, e.g., a region of MUC1-Cthat interacts with galectin-3 or a region of galectin-3 that binds toMUC1-C and/or determine the structures involved in MUC1-C/galectin-3binding. Computer programs such as INSIGHT™ (Accelrys, Burlington,Mass.), GRASP (Anthony Nicholls, Columbia University), Dock (MolecularDesign Institute, University of California at San Francisco), andAuto-Dock (Accelrys) allow for further manipulation and the ability tointroduce new structures.

Compounds can be designed using, for example, computer hardware orsoftware, or a combination of both. However, designing is preferablyimplemented in one or more computer programs executing on one or moreprogrammable computers, each containing a processor and at least oneinput device. The computer(s) preferably also contain(s) a data storagesystem (including volatile and non-volatile memory and/or storageelements) and at least one output device. Program code is applied toinput data to perform the functions described above and generate outputinformation. The output information is applied to one or more outputdevices in a known fashion. The computer can be, for example, a personalcomputer, microcomputer, or work station of conventional design.

Each program is preferably implemented in a high level procedural orobject oriented programming language to communicate with a computersystem. However, the programs can be implemented in assembly or machinelanguage, if desired. In any case, the language can be a compiled orinterpreted language.

Each computer program is preferably stored on a storage media or device(e.g., ROM or magnetic diskette) readable by a general or specialpurpose programmable computer. The computer program serves to configureand operate the computer to perform the procedures described herein whenthe program is read by the computer. The method of the invention canalso be implemented by means of a computer-readable storage medium,configured with a computer program, where the storage medium soconfigured causes a computer to operate in a specific and predefinedmanner to perform the functions described herein.

For example, the computer-requiring steps in a method of designing aninhibitory compound can involve:

(a) inputting into an input device, e.g., through a keyboard, adiskette, or a tape, data (e.g. atomic coordinates) that define thethree-dimensional (3-D) structure of a first molecule (e.g., MUC1-C or apart of MUC1-C) that binds to a second molecule (e.g., galectin-3 or apart thereof) or a molecular complex (e.g., MUC1-C, or a part thereof,bound to galectin-3, or a part thereof), e.g., a region of MUC1-C thatinteracts with galectin-3 (e.g., all or a part of the extracellulardomain of MUC1-C), the region of galectin-3 that binds to MUC1-C (e.g.,a region within the carbohydrate binding domain and/or all or part ofamino acid residues 63-250), or all or a part (e.g., the cytoplasmicdomain) of MUC1-C bound to all or a part (e.g., the carbohydrate bindingdomain and/or amino acid residues 63-250) of galectin-3; and

(b) determining, using a processor, the 3-D structure (e.g., an atomicmodel) of: (i) the site on the first molecule involved in binding to thesecond molecule; or (ii) one or more sites of interaction betweenmolecular components of the molecular complex.

From the information obtained in this way, one skilled in the art willbe able to design and make inhibitory compounds (e.g., peptides,non-peptide small molecules, aptamers (e.g., nucleic acid aptamers) withthe appropriate 3-D structure (see “Methods of Making InhibitoryCompounds and Proteins Useful for the Invention” below).

Moreover, if computer-usable 3-D data (e.g., x-ray crystallographicdata) for a candidate compound are available, the followingcomputer-based steps can be performed in conjunction with computer-basedsteps (a) and (b) described above:

(c) inputting into an input device, e.g., through a keyboard, adiskette, or a tape, data (e.g. atomic coordinates) that define thethree-dimensional (3-D) structure of a candidate compound;

(d) determining, using a processor, the 3-D structure (e.g., an atomicmodel) of the candidate compound;

(e) determining, using the processor, whether the candidate compoundbinds to the site on the first molecule or the one or more sites on themolecular components of the molecular complex; and

(f) identifying the candidate compound as compound that inhibits theinteraction between the first and second molecule or the between themolecular components of the molecular complex.

The method can involve the additional step of outputting to an outputdevice a model of the 3-D structure of the compound. In addition, the3-D data of candidate compounds can be compared to a computer databaseof, for example, 3-D structures (e.g., of MUC1-C, the extracellulardomain of MUC1-C, galectin-3, or the carbohydrate binding domain ofgalectin-3) stored in a data storage system.

Compounds of the invention also may be interactively designed fromstructural information of the compounds described herein using otherstructure-based design/modeling techniques (see, e.g., Jackson, 1997;and Jones et al., 1996).

Compounds and polypeptides of the invention also can be identified by,for example, identifying candidate compounds by computer modeling asfitting spatially and preferentially (i.e., with high affinity) into theappropriate acceptor sites on MUC1-C or galectin-3.

Candidate compounds identified as described above can then be tested instandard cellular or cell-free binding or binding inhibition assaysfamiliar to those skilled in the art. Exemplary assays are describedherein.

A candidate compound whose presence requires at least 2-fold (e.g.,4-fold, 6-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or100,000-fold) more of a given MUC1-C test agent to achieve a definedarbitrary level of binding to a fixed amount of a galectin-3 test agentthan is achieved in the absence of the compound can be useful forinhibiting the interaction between MUC1-C and galectin-3, and thus canbe useful as a cancer therapeutic or prophylactic agent. Alternatively,a candidate compound whose presence requires at least 2-fold (e.g.,2-fold, 4-fold, 6-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or100,000-fold) more of a given galectin-3 test agent to achieve a definedarbitrary level of binding to a fixed amount of a MUC1-C test agent thanis achieved in the absence of the compound can be useful for inhibitingthe interaction between MUC1-C and galectin-3, and thus can be useful asa cancer therapeutic or prophylactic agent.

The 3-D structure of biological macromolecules (e.g., proteins, nucleicacids, carbohydrates, and lipids) can be determined from data obtainedby a variety of methodologies. These methodologies, which have beenapplied most effectively to the assessment of the 3-D structure ofproteins, include: (a) x-ray crystallography; (b) nuclear magneticresonance (NMR) spectroscopy; (c) analysis of physical distanceconstraints formed between defined sites on a macromolecule, e.g.,intramolecular chemical crosslinks between residues on a protein (e.g.,International Patent Application No. PCT/US00/14667, the disclosure ofwhich is incorporated herein by reference in its entirety), and (d)molecular modeling methods based on a knowledge of the primary structureof a protein of interest, e.g., homology modeling techniques, threadingalgorithms, or ab initio structure modeling using computer programs suchas MONSSTER (Modeling Of New Structures from Secondary and TertiaryRestraints) (see, e.g., International Application No. PCT/US99/11913,the disclosure of which is incorporated herein by reference in itsentirety). Other molecular modeling techniques may also be employed inaccordance with this invention [e.g., Cohen et al., 1990; Navia et al.,1992, the disclosures of which are incorporated herein by reference inits entirety]. All these methods produce data that are amenable tocomputer analysis. Other spectroscopic methods that can also be usefulin the method of the invention, but that do not currently provide atomiclevel structural detail about biomolecules, include circular dichroismand fluorescence and ultraviolet/visible light absorbance spectroscopy.A preferred method of analysis is x-ray crystallography. Descriptions ofthis procedure and of NMR spectroscopy are provided below.

X-ray Crystallography

Structure determination by x-ray crystallography is based on thediffraction of x-radiation of a characteristic wavelength by electronclouds surrounding the atomic nuclei in a crystal of a molecule ormolecular complex of interest. The technique uses crystals of purifiedbiological macromolecules or molecular complexes (but these frequentlyinclude solvent components, co-factors, substrates, or other ligands) todetermine near atomic resolution of the atoms making up the particularbiological macromolecule. A prerequisite for solving 3-D structure byx-ray crystallography is a well-ordered crystal that will diffractx-rays strongly. The method directs a beam of x-rays onto a regular,repeating array of many identical molecules so that the x-rays arediffracted from the array in a pattern from which the structure of anindividual molecule can be retrieved. Well-ordered crystals of, forexample, globular protein molecules are large, spherical or ellipsoidalobjects with irregular surfaces. The crystals contain large channelsbetween the individual molecules. These channels, which normally occupymore than one half the volume of the crystal, are filled with disorderedsolvent molecules, and the protein molecules are in contact with eachother at only a few small regions. This is one reason why structures ofproteins in crystals are generally the same as those of proteins insolution.

Methods of obtaining the proteins of interest are described below. Theformation of crystals is dependent on a number of different parameters,including pH, temperature, the concentration of the biologicalmacromolecule, the nature of the solvent and precipitant, as well as thepresence of added ions or ligands of the protein. Many routinecrystallization experiments may be needed to screen all these parametersfor the combinations that give a crystal suitable for x-ray diffractionanalysis. Crystallization robots can automate and speed up the work ofreproducibly setting up a large number of crystallization experiments(see, e.g., U.S. Pat. No. 5,790,421, the disclosure of which isincorporated herein by reference in its entirety).

Polypeptide crystallization occurs in solutions in which the polypeptideconcentration exceeds it's solubility maximum (i.e., the polypeptidesolution is supersaturated). Such solutions may be restored toequilibrium by reducing the polypeptide concentration, preferablythrough precipitation of the polypeptide crystals. Often polypeptidesmay be induced to crystallize from supersaturated solutions by addingagents that alter the polypeptide surface charges or perturb theinteraction between the polypeptide and bulk water to promoteassociations that lead to crystallization.

Crystallizations are generally carried out between 4° C. and 20° C.Substances known as “precipitants” are often used to decrease thesolubility of the polypeptide in a concentrated solution by forming anenergetically unfavorable precipitating depleted layer around thepolypeptide molecules (Weber, 1991). In addition to precipitants, othermaterials are sometimes added to the polypeptide crystallizationsolution. These include buffers to adjust the pH of the solution andsalts to reduce the solubility of the polypeptide. Various precipitantsare known in the art and include the following: ethanol, 3-ethyl-2-4pentanediol, and many of the polyglycols, such as polyethylene glycol(PEG). The precipitating solutions can include, for example, 13-24% PEG4000, 5-41% ammonium sulfate, and 1.0-1.5 M sodium chloride, and a pHranging from 5-7.5. Other additives can include 0.1 M Hepes, 2-4%butanol, 0.1 M or 20 mM sodium acetate, 50-70 mM citric acid, 120-130 mMsodium phosphate, 1 mM ethylene diamine tetraacetic acid (EDTA), and 1mM dithiothreitol (DTT). These agents are prepared in buffers and areadded dropwise in various combinations to the crystallization buffer.

Commonly used polypeptide crystallization methods include the followingtechniques: batch, hanging drop, seed initiation, and dialysis. In eachof these methods, it is important to promote continued crystallizationafter nucleation by maintaining a supersaturated solution. In the batchmethod, polypeptide is mixed with precipitants to achievesupersaturation, and the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane that is placed into a solution containing precipitant.Equilibration across the membrane increases the polypeptide andprecipitant concentrations, thereby causing the polypeptide to reachsupersaturation levels.

In the preferred hanging drop technique (McPherson, 1976), an initialpolypeptide mixture is created by adding a precipitant to a concentratedpolypeptide solution. The concentrations of the polypeptide andprecipitants are such that in this initial form, the polypeptide doesnot crystallize. A small drop of this mixture is placed on a glass slidethat is inverted and suspended over a reservoir of a second solution.The system is then sealed. Typically, the second solution contains ahigher concentration of precipitant or other dehydrating agent. Thedifference in the precipitant concentrations causes the protein solutionto have a higher vapor pressure than the second solution. Since thesystem containing the two solutions is sealed, an equilibrium isestablished, and water from the polypeptide mixture transfers to thesecond solution. This equilibrium increases the polypeptide andprecipitant concentration in the polypeptide solution. At the criticalconcentration of polypeptide and precipitant, a crystal of thepolypeptide may form.

Another method of crystallization introduces a nucleation site into aconcentrated polypeptide solution. Generally, a concentrated polypeptidesolution is prepared and a seed crystal of the polypeptide is introducedinto this solution. If the concentrations of the polypeptide and anyprecipitants are correct, the seed crystal will provide a nucleationsite around which a larger crystal forms.

Yet another method of crystallization is an electrocrystallizationmethod in which use is made of the dipole moments of proteinmacromolecules that self-align in the Helmholtz layer adjacent to anelectrode (see, e.g., U.S. Pat. No. 5,597,457, the disclosure of whichis incorporated herein by reference in its entirety).

Some proteins may be recalcitrant to crystallization. However, severaltechniques are available to the skilled artisan to inducecrystallization. For example, the removal of flexible polypeptidesegments at the amino or carboxyl terminal end of the protein mayfacilitate production of crystalline protein samples. Removal of suchsegments can be done using molecular biology techniques or treatment ofthe protein with proteases such as trypsin, chymotrypsin, or subtilisin.

In diffraction experiments, a narrow and parallel beam of x-rays istaken from the x-ray source and directed onto the crystal to producediffracted beams. The incident primary beams cause damage to both themacromolecule and solvent molecules. The crystal is, therefore, cooled(e.g., to −220° C. to −50° C.) to prolong its lifetime. The primary beammust strike the crystal from many directions to produce all possiblediffraction spots, so the crystal is rotated in the beam during theexperiment. The diffracted spots are recorded on a film or by anelectronic detector. Exposed film has to be digitized and quantified ina scanning device, whereas the electronic detectors feed the signalsthey detect directly into a computer. Electronic area detectorssignificantly reduce the time required to collect and measurediffraction data. Each diffraction beam, which is recorded as a spot onfilm, is defined by three properties: the amplitude, which is measuredfrom the intensity of the spot; the wavelength, which is set by thex-ray source; and the phase, which is lost in x-ray experiments. Allthree properties are needed for all of the diffracted beams in order todetermine the positions of the atoms giving rise to the diffractedbeams. One way of determining the phases is called Multiple IsomorphousReplacement (MIR), which requires the introduction of exogenous x-rayscatterers (e.g., heavy atoms such metal atoms) into the unit cell ofthe crystal. For a more detailed description of MIR, see U.S. Pat. No.6,093,573 (column 15) the disclosure of which is incorporated herein byreference in its entirety.

Atomic coordinates refer to Cartesian coordinates (x, y, and zpositions) derived from mathematical equations involving Fouriersynthesis of data derived from patterns obtained via diffraction of amonochromatic beam of x-rays by the atoms (scattering centers) ofbiological macromolecule of interest in crystal form. Diffraction dataare used to calculate electron density maps of repeating units in thecrystal (unit cell). Electron density maps are used to establish thepositions (atomic coordinates) of individual atoms within a crystal'sunit cell. The absolute values of atomic coordinates convey spatialrelationships between atoms because the absolute values ascribed toatomic coordinates can be changed by rotational and/or translationalmovement along x, y, and/or z axes, together or separately, whilemaintaining the same relative spatial relationships among atoms. Thus, abiological macromolecule (e.g., a protein) whose set of absolute atomiccoordinate values can be rotationally or translationally adjusted tocoincide with a set of prior determined values from an analysis ofanother sample is considered to have the same atomic coordinates asthose obtained from the other sample.

Further details on x-ray crystallography can be obtained from co-pendingU.S. application Ser. No. 10/486,278, U.S. Pat. No. 6,093,573 andInternational Application Nos. PCT/US99/18441, PCT/US99/11913, andPCT/US00/03745. The disclosures of all these patent documents areincorporated herein by reference in their entirety.

NMR Spectroscopy

While x-ray crystallography requires single crystals of a macromoleculeof interest, NMR measurements are carried out in solution under nearphysiological conditions. However, NMR-derived structures are not asdetailed as crystal-derived structures.

While the use of NMR spectroscopy was until relatively recently limitedto the elucidation of the 3-D structure of relatively small molecules(e.g., proteins of 100-150 amino acid residues), recent advancesincluding isotopic labeling of the molecule of interest and transverserelaxation-optimized spectroscopy (TROSY) have allowed the methodologyto be extended to the analysis of much larger molecules, e.g., proteinswith a molecular weight of 110 kDa (Wider, 2000).

NMR uses radio-frequency radiation to examine the environment ofmagnetic atomic nuclei in a homogeneous magnetic field pulsed with aspecific radio frequency. The pulses perturb the nuclear magnetizationof those atoms with nuclei of nonzero spin. Transient time domainsignals are detected as the system returns to equilibrium. Fouriertransformation of the transient signal into a frequency domain yields aone-dimensional NMR spectrum. Peaks in these spectra represent chemicalshifts of the various active nuclei. The chemical shift of an atom isdetermined by its local electronic environment. Two-dimensional NMRexperiments can provide information about the proximity of various atomsin the structure and in three dimensional space. Protein structures canbe determined by performing a number of two- (and sometimes 3- or 4-)dimensional NMR experiments and using the resulting information asconstraints in a series of protein folding simulations.

More information on NMR spectroscopy including detailed descriptions ofhow raw data obtained from an NMR experiment can be used to determinethe 3-D structure of a macromolecule can be found in: Protein NMRSpectroscopy, Principles and Practice, 1996; Gronenborn et al., 1990;and Wider (2000), supra., the disclosures of all of which areincorporated herein by reference in their entirety

Any available method can be used to construct a 3-D model of a region ofMUC1-C and/or galectin-3 from the x-ray crystallographic and/or NMR datausing a computer as described above. Such a model can be constructedfrom analytical data points inputted into the computer by an inputdevice and by means of a processor using known software packages, e.g.,HKL, MOSFILM, XDS, CCP4, SHARP, PHASES, HEAVY, XPLOR, TNT, NMRCOMPASS,NMRPIPE, DIANA, NMRDRAW, FELIX, VNMR, MADIGRAS, QUANTA, BUSTER, SOLVE,O, FRODO, or CHAIN. The model constructed from these data can bevisualized via an output device of a computer, using available systems,e.g., from Silicon Graphics, Evans and Sutherland, SUN, Hewlett Packard,Apple Macintosh, DEC, IBM, or Compaq.

Methods of Making Inhibitory Compounds and Proteins Useful for theInvention

Once the 3-D structure of a protein of interest (MUC1-C or galectin-3),or a binding region-containing fragment thereof, has been establishedusing any of the above methods, a compound that has substantially thesame 3-D structure (or contains a domain that has substantially the samestructure) as the binding region of the protein of interest. Thecompound's structure can be based on the 3-D structure of binding siteof the parent protein (e.g., MUC1-C), the 3-D structure of thecomplementary acceptor site of the protein to which the parent proteinbinds (e.g., galectin-3), or a combination of both. In this context,“has substantially the same 3-D structure” means that the compound bindswith at least the same avidity as the parent protein to the non-parentpartner. The compound can also bind to the non-parent partner with atleast two-fold (at least: three-fold; four-fold; five-fold; six-fold;seven-fold; eight-fold; nine-fold; ten-fold; 20-fold; 50-fold; 100-fold;1,000-fold; 10,000-fold; 100,000-fold; 1,000,000-fold; or evenhigher-fold) greater avidity than the parent protein. One of skill inthe art would know how to test a compound for such an ability.

With the above described 3-D structural data on hand and knowing thechemical structure (e.g., amino acid sequence in the case of a protein)of the protein region of interest, those of skill in the art would knowhow to make compounds with the above-described properties. Such methodsinclude chemical synthetic methods and, in the case of proteins,recombinant methods (see above). For example, cysteine residuesappropriately placed in a compound so as to form disulfide bonds can beused to constrain the compound or a domain of the compound in anappropriate 3-D structure. In addition, in a compound that is apolypeptide or includes a domain that is a polypeptide, one of skill inthe art would know what amino acids to include and in what sequence toinclude them in order to generate, for example, α-helices, β structures,or sharp turns or bends in the polypeptide backbone.

Of particular interest as small molecule compounds are nucleic acidaptamers which are relatively short nucleic acid (DNA, RNA or acombination of both) sequences that bind with high avidity to a varietyof proteins and inhibit the binding to such proteins of ligands,receptors, and other molecules. Aptamers are generally about 25-40nucleotides in length and have molecular weights in the range of about18-25 kDa. Aptamers with high specificity and affinity for targets canbe obtained by an in vitro evolutionary process termed SELEX (systemicevolution of ligands by exponential enrichment) [see, for example, Zhanget al. (2004), the disclosure of which is incorporated herein byreference in its entirety]. For methods of enhancing the stability (byusing nucleotide analogs, for example) and enhancing in vivobioavailability (e.g., in vivo persistence in a subject's circulatorysystem) of nucleic acid aptamers see Zhang et al. (2004) supra and Brodyet al. (2000), the disclosure of which is incorporated herein byreference in its entirety].

While not essential, computer-based methods can be used to design thecompounds of the invention. Appropriate computer programs include: LUDI(Biosym Technologies, Inc., San Diego, Calif.), Aladdin (DaylightChemical Information Systems, Irvine, Calif.); and LEGEND (Nishibata etal., 1985).

The compounds of the invention can include, in addition, to the abovedescribed proteins, one or more domains that facilitate purification(e.g., poly-histidine sequences) or domains that serve to direct thecompound to appropriate target cells (e.g., cancer cells), e.g., ligandsor antibodies (including antibody fragments such as Fab, F(ab′)₂, orsingle chain Fv fragments) specific for cell surface components oftarget cells of the immune system, e.g., MUC1-C, galectin-3, Her2/Neu,or any of a variety of other tumor-associated antigens. Signal sequencesthat facilitate transport of the compounds across biological membranes(e.g., cell membranes and/or nuclear membranes) and/direct them tosubcellular compartments can also be linked (e.g., covalently) to thecompounds. Exemplary signal sequences are described in detail in U.S.Pat. No. 5,827,516, the disclosure of which is incorporated herein byreference in its entirety. All that is required in such multidomaincompounds is that the domain corresponding to the parent inhibitorycompound substantially retains the 3-D structure it would have in theabsence of the additional domains. Conjugation to make such multidomaincompounds can be by chemical methods [e.g., Barrios et al. (1992), thedisclosure of which is incorporated herein by reference in itsentirety]. Where the compound is a peptide, it can be produced as partof a recombinant protein, such as one that self-assembles intovirus-sized particles (e.g., U.S. Pat. No. 4,918,166, the disclosure ofwhich is incorporated herein by reference in its entirety) that displaycandidate binding peptides on the surface.

Compounds of the invention that are peptides also include thosedescribed above, but modified for in vivo use by the addition, at theamino- and/or carboxyl-terminal ends, of a blocking agent to facilitatesurvival of the relevant polypeptide in vivo. This can be useful inthose situations in which the peptide termini tend to be degraded byproteases prior to cellular uptake. Such blocking agents can include,without limitation, additional related or unrelated peptide sequencesthat can be attached to the amino and/or carboxyl terminal residues ofthe peptide to be administered. This can be done either chemicallyduring the synthesis of the peptide or by recombinant DNA technology bymethods familiar to artisans of average skill

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptide compounds can be covalently or noncovalentlycoupled to pharmaceutically acceptable “carrier” proteins prior toadministration.

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of compounds of the invention that arepeptides. Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to inhibit the interaction between MUC1-C andgalectin-3. Peptidomimetic compounds can have additional characteristicsthat enhance their in vivo utility, such as increased cell permeabilityand prolonged biological half-life. The peptidomimetics typically have abackbone that is partially or completely non-peptide, but with sidegroups that are identical to the side groups of the amino acid residuesthat occur in the peptide on which the peptidomimetic is based. Severaltypes of chemical bonds, e.g., ester, thioester, thioamide, retroamide,reduced carbonyl, dimethylene and ketomethylene bonds, are known in theart to be generally useful substitutes for peptide bonds in theconstruction of protease-resistant peptidomimetics.

The proteins (MUC1-C or galectin-3) used for designing compounds of theinvention and all other methods described herein can be purified fromnatural sources (e.g., from tissues such as pancreas, liver, lung,breast, skin, spleen, ovary, testis, muscle, joint tissue, neuraltissue, gastrointestinal tissue or tumor tissue (e.g., breast cancer orcolon cancer tissue), or body fluids such as blood, serum, or urine).Smaller peptides (fewer than 100 amino acids long) and other non-proteincompounds of the invention can be conveniently synthesized by standardchemical means known to those in the art. In addition, both polypeptidesand peptides can be manufactured by standard in vitro recombinant DNAtechniques and in vivo transgenesis using nucleotide sequences encodingthe appropriate polypeptides or peptides. Methods well-known to thoseskilled in the art can be used to construct expression vectorscontaining relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example, thetechniques described in Sambrook et al. (1989), and Ausubel et al.(1989).

For the structural (e.g., x-ray crystallographic and NMR) analysesdescribed above, it is generally required that proteins, or fragmentsthereof, be highly purified. Methods for purifying biologicalmacromolecules (e.g., proteins) are known in the art. The degree ofpurity of proteins can be measured by any appropriate method, e.g.,column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

MUC1-C and galectin-3 used for the above analyses can be of anymammalian species, e.g., humans, non-human primates (e.g., monkeys,baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs,cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.

Methods of Inhibiting Binding of MUC1-C to Galectin-3 in or on a Cell

The invention features methods of inhibiting binding of MUC1-C togalectin-3 in or on a cell. The method involves introducing to thesurface of, or into, the cell a compound that inhibits the binding ofgalectin-3 to MUC1-C (e.g., to the MUC1 ED). Prior to introduction ofthe compound to or into the cell, the cell (or another cancer cell fromthe subject from which the cell to be treated was obtained) canoptionally be tested for MUC1 expression. This can be done by testingfor expression of either MUC1 protein or MUC 1 mRNA by any of a widevariety of methods known in the art.

The compound can be one identified by the methods described above.Examples of appropriate compounds include the ED of human MUC1-C,peptide fragments of the ED of MUC1-C that bind to galectin-3, andfragments of galectin-3 that bind MUC1-C. An appropriate fragment of thehuman MUC1-C can be one containing or consisting of all or part of aminoacids 1-58 of SEQ ID NO:1. Other useful inhibitory compounds can bemolecules that contain or consist of all or part of amino acids 63-250or 117-244 of galectin-3 (SEQ ID NO:2).

Peptide inhibitory compounds can contain up to 50 (e.g., one, two,three, four, five, six, seven, eight, nine, ten, 12, 15, 18, 20, 25, 30,35, 40, 45, or 50) MUC1 or galectin-3 residues or unrelated residues oneither end or on both ends of the MUC1 or galectin-3 inhibitorysegments.

Any MUC1 or galectin-3 peptides to be used as inhibitor compounds canoptionally have any phosphorylation-susceptible amino acid residuesphosphorylated, e.g., S6 of SEQ ID NO:2. Any MUC1-C peptides to be usedas inhibitor compounds can optionally be glycosylated on a residuecorresponding to N36 of SEQ ID NO:1.

MUC1 peptide fragments useful as inhibitory compounds (or otherinhibitory compounds (e.g., galectin-3-specific antibodies or antibodyfragments) that act by binding galectin-3) will have substantially noMUC1 agonist activity, i.e., they will substantially lack the effects ofMUC1 described herein that result from binding of MUC1-C to galectin-3.Compounds having substantially no MUC1 agonist activity are those havingless than 20% (e.g., less than: 10%; 5%; 2%; 1%; 0.5%; 0.2%; 0.1%;0.01%; 0.001%; or 0.0001%) of the ability of MUC1-C to increase the mRNAlevel of galectin-3.

Similarly, galectin-3 peptide fragment compounds will have substantiallynone of the expression-enhancing activity of galectin-3 on thegalectin-3 gene that occurs in the presence of nonlimiting amounts ofMUC1. Compounds having substantially none of the activity of galectin-3that occurs in the presence of nonlimiting amounts of MUC1, have lessthan 20% (e.g., less than: 10%; 5%; 2%; 1%; 0.5%; 0.2%; 0.1%; 0.01%;0.001%; or 0.0001%) of galectin-3 to increase the mRNA level ofgalectin-3. Methods of designing, making, and testing such compounds forthe appropriate binding-inhibitory activity are known to those in theart.

In addition, the inhibitory compounds can be antibodies, orantigen-binding antibody fragments, specific for MUC1 or galectin-3.Such antibodies will generally bind to, or near to: (a) the region ofMUC1 to which galectin-3 binds; (b) or the region on galectin-3 to whichMUC1 binds. However, as indicated above, the compounds can also actallosterically and so they can also bind to the three proteins atpositions other than, and even remote from, the binding sites for MUC1(on galectin-3) and on galectin-3 (for MUC1). As used throughout thepresent application, the term “antibody” refers to a whole antibody(e.g., IgM, IgG, IgA, IgD, or IgE) molecule that is generated by any oneof a variety of methods that are known in the art. The antibody can bemade in or derived from any of a variety of species, e.g., humans,non-human primates (e.g., monkeys, baboons, or chimpanzees), horses,cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils,hamsters, rats, and mice.

The antibody can be a purified or a recombinant antibody. Also usefulfor the invention are antibody fragments and chimeric antibodies andhumanized antibodies made from non-human (e.g., mouse, rat, gerbil, orhamster) antibodies. As used herein, the term “antibody fragment” refersto an antigen-binding fragment, e.g., Fab, F(ab′)₂, Fv, and single chainFv (scFv) fragments. An scFv fragment is a single polypeptide chain thatincludes both the heavy and light chain variable regions of the antibodyfrom which the scFv is derived. In addition, diabodies (Poljak, 1994;Hudson et al., 1999, the disclosures of both of which are incorporatedherein by reference in their entirety) and intrabodies (Huston et al.,2001; Wheeler et al., 2003; Stocks, 2004, the disclosures of all ofwhich are incorporated herein by reference in their entirety] can beused in the methods of the invention).

Antibody fragments that contain the binding domain of the molecule canbe generated by known techniques. For example: F(ab′)₂ fragments can beproduced by pepsin digestion of antibody molecules; and Fab fragmentscan be generated by reducing the disulfide bridges of F(ab′)₂ fragmentsor by treating antibody molecules with papain and a reducing agent. See,e.g., National Institutes of Health, 1991) the disclosure of which isincorporated herein by reference in their entirety. scFv fragments canbe produced, for example, as described in U.S. Pat. No. 4,642,334, thedisclosure of which is incorporated herein by reference in its entirety.

Chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example, using methodsdescribed in Robinson et al., International Patent PublicationPCT/US86/02269; Akira et al., European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., PCT ApplicationWO 86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al., 1988; Liu et al.,1987; Sun et al., 1987; Nishimura et al., 1987; Wood et al., 1985; Shawet al., 1988; Morrison, 1985; Oi et al., 1986; Winter, U.S. Pat. No.5,225,539; Jones et al., 1986; Veroeyan et al., 1988; and Beidler etal., 1988). The disclosures of all these articles and patent documentsare incorporated herein by reference in their entirety.

Other compounds useful to inhibit the binding of MUC1-C to galectin-3include β-galactoside carbohydrates (e.g., lactose) and carbohydrateanalogs that bind to galectin-3 (see, e.g., Ahmad et al., 2004).

Also useful for inhibiting the binding of MUC1-C to galectin-3 arecompounds capable of deglycosylating MUC1 (e.g., deglycosylatingenzymes) or capable of inhibiting the glycosylation (e.g.,N-glycosylation) of MUC1 (e.g., tunicamycin).

Compounds that inhibit the binding of galectin-3 to the extracellulardomain of MUC1-C can also be useful for inhibiting the association ofMUC1 and EGFR in cells (e.g., cancer cells) that express MUC1.

Cells to which the method of the invention can be applied includegenerally any cell that expresses MUC1. Such cells include normal cells,such as any normal epithelial cell, or a cancer cell, whoseproliferation it is desired to inhibit. An appropriate cancer cell canbe a breast cancer, lung cancer, colon cancer, pancreatic cancer, renalcancer, stomach cancer, liver cancer, bone cancer, hematological cancer(e.g., leukemia or lymphoma), neural tissue cancer, melanoma, ovariancancer, testicular cancer, prostate cancer, cervical cancer, vaginalcancer, or bladder cancer cell. In addition, the methods of theinvention can be applied to a wide range of species, e.g., humans,non-human primates (e.g., monkeys, baboons, or chimpanzees), horses,cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils,hamsters, rats, and mice. It will be appreciated that proteins (e.g.,galectin-3 and MUC-1), fragments thereof, and other compounds recitedherein as useful for the invention can be of any of these species.

The methods can be performed in vitro, in vivo, or ex vivo. In vitroapplication of appropriate compounds can be useful, for example, inbasic scientific studies of tumor cell biology, e.g., studies on themechanism of action of MUC1 and/or galectin-3 in promoting tumor cellgrowth, including survival. In addition, the compounds that areinhibitory can be used as “positive controls” in methods to identifyadditional compounds with inhibitory activity (see above). In such invitro methods, cells expressing MUC1 and galectin-3 can be incubated forvarious times with the inhibitory compound(s) at a variety ofconcentrations. Other incubation conditions known to those in art (e.g.,temperature, or cell concentration) can also be varied. Inhibition ofbinding can be tested by methods such as those disclosed herein.

The methods of the invention will preferably be in vivo or ex vivo.

Compounds that inhibit binding between MUC 1 and galectin-3 aregenerally useful as cancer cell (e.g., breast cancer cell)survival-inhibiting and/or cell cycle-arresting therapeutics orprophylactics. They can be administered to mammalian subjects (e.g.,human breast cancer patients) alone or in conjunction with other drugsand/or radiotherapy. The compounds can also be administered to subjectsthat are genetically and/or due to, e.g., physiological and/orenvironmental factors, susceptible to cancer (e.g., subjects with afamily history of cancer (e.g., breast cancer), subjects with chronicinflammation or subject to chronic stress, or subjects that are exposedto natural or non-natural environmental carcinogenic conditions (e.g.,excessive exposure to sunlight, industrial carcinogens, or tobaccosmoke)). As used herein, a compound that is “therapeutic” is a compoundthat causes a complete abolishment of the symptoms of a disease or adecrease in the severity of the symptoms of the disease. “Prevention”means that symptoms of the disease (e.g., cancer) are essentiallyabsent. As used herein, “prophylaxis” means complete prevention of thesymptoms of a disease, a delay in onset of the symptoms of a disease, ora lessening in the severity of subsequently developed disease symptoms.

When the methods are applied to subjects with cancer, prior toadministration of a compound, the cancer can optionally be tested forMUC1 expression and/or galectin-3 expression (e.g., protein or mRNAexpression) by methods known in the art. In this way, subjects can beidentified as having a MUC1- or galectin-3-expressing cancer. Suchmethods can be performed in vitro on cancer cells obtained from asubject (e.g., by biopsy). Alternatively, in vivo imaging techniquesusing, for example, radiolabeled antibodies specific for MUC1 orgalectin-3 can be performed. In addition, body fluids (e.g., blood orurine) from subjects with cancer can be tested for elevated levels ofMUC1 or galectin-3 protein or protein fragments.

A cell from a subject (e.g., a suspected cancer cell) can be tested forgalectin-3 expression (e.g., protein or mRNA expression) and/orexpression of a MUC1-galectin-3 protein complex by methods known in theart. An elevated level of galectin-3 or a MUC1-galectin-3 complex (e.g.,compared to a reference standard or another cell of the subject) is anindication that the cell is a cancer cell. These methods can be used indiagnosis of cancers.

Cell-Based Methods

In some instances, cells can be tested for MUC1 expression and/orgalectin-3 expression (e.g., protein or mRNA expression) by methodsknown in the art. Such methods can be performed in vitro on cancer cellsobtained from a subject (e.g., by biopsy). Alternatively, in vivoimaging techniques using, for example, radiolabeled antibodies specificfor MUC1 or galectin-3 can be performed.

Cells (e.g., in vitro or in vivo) that express MUC1 can be moresusceptible to apoptosis when galectin-3 is dephosphorylated. Apoptosiscan be promoted in such a cell by contacting the cell with a compoundthat inhibits phosphorylation of galectin-3 (e.g., on Serb) by caseinkinase 1. Exemplary compounds are described (Chijiwa et al., 1989;Meggio et al., 1990; Mashhoon et al., 2000; and Rena et al., 2004).

In vivo Approaches

In one in vivo approach, a compound that inhibits binding of MUC1 togalectin-3 is administered to a subject. Generally, the compounds of theinvention will be suspended in a pharmaceutically-acceptable carrier(e.g., physiological saline) and administered orally or injectedintravenously, subcutaneously, intramuscularly, intrathecally,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily. They can also bedelivered directly to tumor cells, e.g., to a tumor or a tumor bedfollowing surgical excision of the tumor, in order to kill any remainingtumor cells. The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.0001 mg/kg—100 mg/kg.Wide variations in the needed dosage are to be expected in view of thevariety of compounds available and the differing efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2-, 3-, 4-,6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of thepolypeptide in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

Alternatively, where an inhibitory compound is a polypeptide, apolynucleotide containing a nucleic acid sequence encoding thepolypeptide can be delivered to appropriate cells in a mammal.Expression of the coding sequence can be directed to any cell in thebody of the subject. However, expression will preferably be directed tocells in the vicinity of the tumor cells whose proliferation it isdesired to inhibit. Expression of the coding sequence can be directed tothe tumor cells themselves. This can be achieved by, for example, theuse of polymeric, biodegradable microparticle or microcapsule deliverydevices known in the art.

Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors can be incorporated alone intothese delivery vehicles or co-incorporated with tissue-specific ortumor-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano etal., (1995, the disclosure of which is incorporated herein by referencein its entirety). Alternatively, tissue specific targeting can beachieved by the use of tissue-specific transcriptional regulatoryelements (TRE) which are known in the art. Delivery of “naked DNA”(i.e., without a delivery vehicle) to an intramuscular, intradermal, orsubcutaneous site is another means to achieve in vivo expression.

In the relevant polynucleotides (e.g., expression vectors), the nucleicacid sequence encoding the polypeptide of interest with an initiatormethionine and optionally a targeting sequence is operatively linked toa promoter or enhancer-promoter combination. Short amino acid sequencescan act as signals to direct proteins to specific intracellularcompartments. Such signal sequences are described in detail in U.S. Pat.No. 5,827,516, the disclosure of which is incorporated herein byreference in its entirety.

Enhancers provide expression specificity in terms of time, location, andlevel. Unlike a promoter, an enhancer can function when located atvariable distances from the transcription initiation site, provided apromoter is present. An enhancer can also be located downstream of thetranscription initiation site. To bring a coding sequence under thecontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. Promoters of interest include but are not limited to thecytomegalovirus hCMV immediate early gene, the early or late promotersof SV40 adenovirus, the lac system, the trp system, the TAC system, theTRC system, the major operator and promoter regions of phage A, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase, the promoters of acid phosphatase, and the promoters of theyeast α-mating factors, the adenoviral E1b minimal promoter, or thethymidine kinase minimal promoter. The DF3 enhancer can be particularlyuseful for expression of an inhibitory compound in cells that naturallyexpress MUC1, for example, normal epithelial cells or malignantepithelial cells (carcinoma cells), e.g., breast cancer cells [see U.S.Pat. Nos. 5,565,334 and 5,874,415, the disclosures of which areincorporated herein by reference in their entirety]. The coding sequenceof the expression vector is operatively linked to a transcriptionterminating region.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles that are suitable for administration to a human,e.g., physiological saline or liposomes. A therapeutically effectiveamount is an amount of the polynucleotide that is capable of producing amedically desirable result (e.g., decreased proliferation of cancercells) in a treated animal. As is well known in the medical arts, thedosage for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages will vary, but apreferred dosage for administration of polynucleotide is fromapproximately 10⁶ to approximately 10¹² copies of the polynucleotidemolecule. This dose can be repeatedly administered, as needed. Routes ofadministration can be any of those listed above.

Ex Vivo Approaches

An ex vivo strategy can involve transfecting or transducing cells,optionally but not necessarily obtained from the subject to be treated,with a polynucleotide encoding a polypeptide that inhibit binding ofMUC1 to galectin-3. The transfected or transduced cells are thenreturned to the subject. The cells can be any of a wide range of typesincluding, without limitation, hemopoietic cells (e.g., bone marrowcells, macrophages, monocytes, dendritic cells, T cells, or B cells),fibroblasts, epithelial cells, endothelial cells, keratinocytes, ormuscle cells. Such cells act as a source of the inhibitory polypeptidefor as long as they survive in the subject. Alternatively, tumor cells,preferably obtained from the subject but potentially from an individualother than the subject, can be transfected or transformed by a vectorencoding the inhibitory polypeptide. The tumor cells, preferably treatedwith an agent (e.g., ionizing irradiation) that ablates theirproliferative capacity, are then introduced into the patient, where theysecrete the polypeptide.

The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the polypeptide that inhibits inhibit binding of MUC1 togalectin-3 or glycosylation of MUC1. These methods are known in the artof molecular biology. The transduction step is accomplished by anystandard means used for ex vivo gene therapy, including calciumphosphate, lipofection, electroporation, viral infection, and biolisticgene transfer. Alternatively, liposomes or polymeric microparticles canbe used. Cells that have been successfully transduced can be selected,for example, for expression of the coding sequence or of a drugresistance gene. The cells may then be lethally irradiated (if desired)and injected or implanted into the subject or another subject.

Methods of Inhibiting Expression of MUC1 or Galectin-3 in a Cell

Also included in the invention are methods of inhibiting expression ofMUC1 or galectin-3 in cells. The method involves introducing into a cellan inhibiting oligonucleotide such as: (a) an antisense oligonucleotidethat hybridizes to a MUC1 or galectin-3 transcript, the antisenseoligonucleotide inhibiting expression of MUC1 or galectin-3 in the cell,(b) a MUC1 or galectin-3 small interference RNA (siRNA), or (c) a MUC1or galectin-3 microRNA (miRNA).

The cells and species to which these methods are applied are the same asthose recited above in “Methods of Inhibiting Binding of MUC1 toGalectin-3 in or on a Cell.”

Prior to introduction of an inhibiting oligonucleotide into the cell,the cell (or another cancer cell from the subject from which the cell tobe treated was obtained) can optionally be tested for expression of MUC1or galectin-3 as described above.

Antisense oligonucleotides hybridize to MUC1 or galectin-3 transcriptsand have the effect in the cell of inhibiting expression of the proteintranslated from the relevant transcripts or of a protein whoseexpression is regulated by the first protein. Thus, for example,expression of galectin-3 can be inhibited by inhibition of expression ofMUC1. Inhibiting expression of MUC1 or galectin-3 in a cell can inhibitcancer cell survival as well as other cancer-enhancing activitiesassociated with MUC1 or galectin-3 expression, e.g., cancer cellproliferation and defective adhesion of cancer cells to neighboringcells. The method can thus be applied to the therapy of cancer,including metastasis.

Antisense compounds are generally used to interfere with proteinexpression either by, for example, interfering directly with translationof a target mRNA molecule, by RNAse-H-mediated degradation of the targetmRNA, by interference with 5′ capping of mRNA, by prevention oftranslation factor binding to the target mRNA by masking of the 5′ cap,or by inhibiting of mRNA polyadenylation. The interference with proteinexpression arises from the hybridization of the antisense compound withits target mRNA. A specific targeting site on a target mRNA of interestfor interaction with a antisense compound is chosen. Thus, for example,for modulation of polyadenylation a preferred target site on an mRNAtarget is a polyadenylation signal or a polyadenylation site. Fordiminishing mRNA stability or degradation, destabilizing sequences arepreferred target sites. Once one or more target sites have beenidentified, oligonucleotides are chosen which are sufficientlycomplementary to the target site (i.e., hybridize sufficiently wellunder physiological conditions and with sufficient specificity) to givethe desired effect.

The miR-322 miRNA has been identified herein as an inhibitor ofgalectin-3 mRNA stability. The human miR-322 miRNA nucleotide sequenceis AAACGUGAGGCGCUGCUAUA (SEQ ID NO:4). Mouse and rat miR-322 miRNAs havealso been identified (see FIG. 3B). Expression of miR-322 miRNA isinhibited by MUC1-C (e.g., N-glycosylated MUC1-C).

With respect to this invention, the term “oligonucleotide” refers to anoligomer or polymer of RNA, DNA, a combination of the two, or a mimeticof either. The term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars, and covalent internucleoside(backbone) linkages. The normal linkage or backbone of RNA and DNA is a3′ to 5′ phosphodiester bond. The term also refers however tooligonucleotides composed entirely of, or having portions containing,non-naturally occurring components which function in a similar manner tothe oligonucleotides containing only naturally-occurring components.Such modified substituted oligonucleotides are often preferred overnative forms because of desirable properties such as, for example,enhanced cellular uptake, enhanced affinity for target sequence, andincreased stability in the presence of nucleases. In the mimetics, thecore base (pyrimidine or purine) structure is generally preserved but(1) the sugars are either modified or replaced with other componentsand/or (2) the inter-nucleobase linkages are modified. One class ofnucleic acid mimetic that has proven to be very useful is referred to asprotein nucleic acid (PNA). In PNA molecules the sugar backbone isreplaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The bases are retained and are bounddirectly to the aza nitrogen atoms of the amide portion of the backbone.PNA and other mimetics useful in the instant invention are described indetail in U.S. Pat. No. 6,210,289, the disclosure of which isincorporated herein by reference in its entirety.

The antisense oligomers to be used in the methods of the inventiongenerally comprise about 8 to about 100 (e.g., about 14 to about 80 orabout 14 to about 35) nucleobases (or nucleosides where the nucleobasesare naturally occurring).

The antisense oligonucleotides can themselves be introduced into a cellor an expression vector containing a nucleic sequence (operably linkedto a TRE) encoding the antisense oligonucleotide can be introduced intothe cell. In the latter case, the oligonucleotide produced by theexpression vector is an RNA oligonucleotide and the RNA oligonucleotidewill be composed entirely of naturally occurring components.

The methods of the invention can be in vitro or in vivo. In vitroapplications of the methods can be useful, for example, in basicscientific studies on cancer cell growth, survival, and metastasis.Moreover, since studies described herein show that inhibiting MUC1 orgalectin-3 expression resulted in enhanced activity of genotoxicchemotherapeutic agents, they can be used in screening, for example,genotoxic compounds for cancer chemotherapeutic efficacy. In such invitro methods, appropriate cells (e.g., those expressing MUC1 orgalectin-3), can be incubated for various lengths of time with (a) theantisense oligonucleotides or (b) expression vectors containing nucleicacid sequences encoding the antisense oligonucleotides at a variety ofconcentrations. Other incubation conditions known to those in art (e.g.,temperature or cell concentration) can also be varied. Inhibition ofMUC1 or galectin-3 expression or cancer cell survival can be tested bymethods known to those in the art, e.g., methods such as those disclosedherein. However, the methods of the invention will preferably be invivo.

The antisense methods are generally useful for cancer cell (e.g., breastcancer cell) survival-inhibiting, proliferation-inhibiting, and/ormetastasis-inhibiting therapy. They can be administered to mammaliansubjects (e.g., human breast cancer patients) alone or in conjunctionwith other drugs and/or radiotherapy. Prior to administration of anantisense oligonucleotide to a subject with cancer, the subject can beidentified as having a cancer in which the cancer cells express MUC1.Methods for testing this are described above. Doses, formulations,routes of administration, vectors, and targeting are as described for invivo approaches to inhibiting the binding of MUC1 to MUC-1-binders in acell. Naturally, the antisense oligonucleotides and expression vectorscontaining nucleic acid sequences encoding the antisenseoligonucleotides will preferably be targeted to cells whose survivaland/or growth arrest it is desired to inhibit.

The invention also includes both in vivo and in vitro methods ofinhibiting expression of MUC1 that involve the use of compounds (smallinterference (si)RNA, miRNA, or other small molecules) that inhibittranscription of the MUC1 or galectin-3 gene, stability of the MUC1 orgalectin-3 mRNA, and/or translation of MUC1 or galectin-3 mRNA bynon-antisense mechanisms. In vitro methods are essentially the same asthose described above for antisense methods. In vivo methods involveadministration to any of the subjects and by any of the doses and routesdisclosed herein. Subjects will preferably be those with cancer, e.g.,human cancer patients. Doses, formulations, routes of administration,vectors, and targeting are as described for in vivo antisenseapproaches. While the invention is not limited by any particularmechanism of action, such compounds can be those that act by eitherinhibiting the binding and/or the activity of transcription factors orby altering the stability of MUC1 or galectin-3 mRNA.

Double-stranded small interference RNA (siRNA) homologous to MUC1 orgalectin-3 DNA can be used to reduce expression of MUC1 or galectin-3 incancer cells. See, e.g., Fire et al. (1998); Romano and Masino (1992);Cogoni et al. (1996); Cogoni and Masino (1999); Misquitta and Paterson(1999); and Kennerdell and Carthew (1998). The disclosures of all thesearticles are incorporated herein by reference in their entirety.

The sense and anti-sense RNA strands of siRNA can be individuallyconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, each strand can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecule or to increase the physical stability of theduplex formed between the sense and anti-sense strands, e.g.,phosphorothioate derivatives and acridine substituted nucleotides. Someof the nucleotides (e.g., the terminal (either terminus) one, two,three, or four nucleotides) can also be deoxyribonucleotides. The senseor anti-sense strand can also be produced biologically using anexpression vector into which a target MUC1 or galectin-3 sequence(full-length or a fragment) has been subcloned in a sense or anti-senseorientation. The sense and anti-sense RNA strands can be annealed invitro before delivery of the dsRNA to cells. Alternatively, annealingcan occur in vivo after the sense and anti-sense strands aresequentially delivered to tumor cells and/or tumor-infiltratingleukocytes.

Of interest are siRNA that target, for example, the MUC1 gene sequence5′-AAGTTCAGTGCCCAGCTCTAC-3′ (SEQ ID NO:9). The sense strand of such asiRNA could have the sequence 5′-GUUCAGUGCCCAGCUCUACUU-3′ (SEQ ID NO:10)or 5′-GUUCAGUGCCCAGCUCUACdTdT-3′ (SEQ ID NO:11) and the antisense strandcould have the sequence 5′-GUAGAGCUGGGCACUGAACUU-3′ (SEQ ID NO:12) or5′-GUAGAGCUGGGCACUGAACdTdT-3′ (SEQ ID NO:13). Also useful is a siRNAcontaining the MUC1 sequence 5′-GGUACCAUCAAUGUCCACG-3′ (sense strand;SEQ ID NO:14) and 5′-CGUGGACAUUGAUGGUACC-3′ (antisense strand; SEQ IDNO:15).

Double-stranded siRNA interference can also be achieved by introducinginto cells (e.g. cancer cells) a polynucleotide from which sense andanti-sense RNAs can be transcribed under the direction of separatepromoters, or a single RNA molecule containing both sense and anti-sensesequences can be transcribed under the direction of a single promoter.

In any of the above methods of inhibiting the interaction between MUC1and galectin-3 and of inhibiting expression of MUC1 and/or galectin-3,one or more agents (e.g., two, three, four, five, six, seven, eight,nine, ten, 11, 12, 15, 18, 20, 25, 30, 40, 50, 60, 70, 80, 100, or more)including, for example, inhibitory compounds, antisenseoligonucleotides, siRNA, miRNA, drugs, aptamers, or other smallmolecules (or vectors encoding them), can be used.

The above methods of inhibiting MUC1 expression can further be used toinhibit expression of galectin-3 in a cell, e.g., a cancer cell (e.g., abreast cancer cell). See Examples 2 and 3.

The above in vivo and ex vivo methods of inhibiting interactions betweenMUC1 and galectin-3 and inhibiting expression of MUC1 and/or galectin-3can be used in conjunction with any of a variety of other cancertherapeutic/prophylactic regimens (e.g., chemotherapeutic,radiotherapeutic, biotherapeutic/prophylactic, andimmunotherapeutic/prophylactic regimens). Of particular interest areregimens involving genotoxic (DNA-damaging) agents. Such agents includevarious forms of ionizing and non-ionizing radiation and a variety ofchemotherapeutic compounds.

Non-ionizing radiation includes, for example, ultra-violet (UV)radiation, infra-red (IR) radiation, microwaves, and electronicemissions. The radiation employed in the methods of the invention ispreferably ionizing radiation. As used herein, “ionizing radiation”means radiation composed of particles or photons that have sufficientenergy or can produce sufficient energy by atomic nuclear interactionsto produce ionization (gain or loss of electrons) of an atom. Ionizingradiation thus includes, without limitation, α-radiation, β-radiation,γ-radiation, or x-radiation. A preferred radiation is x-radiation.

Ionizing radiation causes DNA damage and cell killing generally inproportion to the dose administered. It has been indicated that themultiple biological effects induced by ionizing radiation are due eitherto the direct interaction of the radiation with DNA or to the formationof free radical species which lead to damage of DNA. These effectsinclude gene mutations, malignant transformation, and cell killing.

External and internal means for delivering ionizing radiation to atarget tissue or cell are known in the art. External sources include βor γ sources or linear accelerators and the like. Alternatively,ionizing radiation may be delivered, for example, by administering aradiolabeled antibody that is capable of binding to a molecule expressedon the surface of a carcinoma (e.g., MUC1 or Her2/neu) to a subject, orby implantation of radiation-emitting pellets in or near the tumor(brachytherapy).

The amount of radiation (e.g., ionizing radiation) needed to kill agiven cell generally depends upon the nature of the cell. As usedherein, an “effective dose” of radiation means a dose of radiation thatproduces cell damage or death when given in conjunction with anadenoviral vector of the invention. Means of determining an effectivedose are known in the art. X-radiation dosages range from daily doses of50 to 200 roentgens for prolonged periods of time (e.g., 6-8 weeks oreven longer) to single doses of 2,000 to 6,000 roentgens. Dosages foradministered radioisotopes vary widely, and depend on the half-life ofthe isotope, the strength and type of radiation emitted, and the degreeof uptake by the target cells.

As used herein, “chemotherapeutic agents” are chemical compounds thatenter cells and damage DNA. Thus, they can be compounds which, forexample, directly cross-link DNA (e.g., cisplatin (CDDP) and otheralkylating agents), intercalate into DNA, or interfere with DNAreplication, mitosis, or chromosomal segregation, e.g., adriamycin (alsoknown as doxorubicin), VP-16 (also known as etoposide), verapamil,podophyllotoxin, and the like. These compounds are widely used in thetreatment of carcinomas. Chemotherapeutic agents useful in the methodsof the invention include, without limitation, cisplatin, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide,verapamil, podophyllotoxin, tamoxifen, taxol, transplatinum,5-fluorouracil, vincristin, vinblastin, methotrexate, or any analog orderivative of these that is effective in damaging DNA.

Routes of administration are the same as those disclosed herein forinteraction-inhibiting and expression-inhibiting compounds. Doses andfrequency of administration vary widely according to all the variableslisted above for administration of interaction-inhibiting andexpression-inhibiting compounds. For example, adriamycin can beadministered by bolus intravenous injection at doses in the range of25-75 mg/m² and etoposide can be administered intravenously or orally atdoses in the range of 35-100 mg/m². Methods of determining optimalparameters of administration are well known in the art.

Combination treatments can include administration of one or more (e.g.,one, two, three, four, five, six, seven, eight, nine or ten)interaction-inhibiting and/or expression-inhibiting compounds of theinvention, and one or more (e.g., one, two, three, four, five, six,seven, eight, nine or ten) radiation modalities, and/or one or more(e.g., one, two, three, four, five, six, seven, eight, nine or ten)chemotherapeutic agents. The interaction-inhibiting andexpression-inhibiting compounds, radiation treatments andchemotherapeutic agents can be given in any order and frequency. Theycan be given simultaneously or sequentially. Treatment with any one ofthe modalities (interaction-inhibiting and expression-inhibitingcompounds, radiation, or chemotherapeutic agents) can involve single ormultiple (e.g., two, three, four, five, six, eight, nine, ten, 12, 15,20, 30, 40, 50, 60, 80, 100, 200, 300, 500, or more) administrationsseparated by any time period found to be optimal in terms of therapeuticbenefit. Multiple administrations can be separated by one to 23 hours, aday, two, three days, four days, five days, six days, seven days, eight,ten days, twelve days, two weeks, three weeks four weeks, a month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, eleven months, a year,one and one half of a year, two years, three years, five years, or tenyears. Administrations can be continued for as long as the subject isneed of the treatment, e.g., any of the of the above time intervals, andcan be for the life of the subject. Administrations can be, for example,once a week for the life-time of the subject. When administrations ofany or all of the modalities are multiple, the course of any one can besimultaneous with, overlapping with, or consequent to the course(s) ofthe other(s).

EXAMPLES

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

Cell Culture.

Human breast cancer (ZR-75-1, BT549) and lung cancer (CRL-5985,CRL-5909) cells were grown in RPMI1640 medium supplemented with 10%fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/mlstreptomycin. The human breast cancer (MD-MB-431, MCF-7), prostatecancer (LnCAP, DU145, PC3), lung cancer (A549), kidney epithelial (293)and Chinese hamster ovary (CHO-K1) cells were cultured in Dulbecco'sModified Eagle's medium (DMEM) supplemented with 10% FBS, antibioticsand 1 mM L-glutamine. The glycosylation deficient CHO-Lec2 and CHO-Lec8cells (ATCC, Manassas, Va.) were grown in alpha minimum essential mediumcontaining 10% FBS and antibiotics. In certain studies, cells werecultured in medium with 0.1% FBS for 24 hours and then stimulated with100 ng/ml epidermal growth factor (EGF) (Calbiochem-Novabiochem, LaJolla, Calif.) for 5 minutes at 37° C.

Immunoblotting and Precipitates.

Lysates were prepared from subconfluent cells as described (Li et al.,2001). Immunoblot analysis was performed with antibodies specific forMUC1-C (Ab5; Neomarkers, Fremont, Calif.), galectin-3 (Abcam, Cambridge,Mass.), β-actin (Sigma-Aldrich, St. Louis, Mo.), epidermal growth factorreceptor (EGFR) (Santa Cruz Biotechnology, Santa Cruz, Calif.), andMUC1-N (DF3) (Ren et al., 2004). Immune complexes were prepared asdescribed with antibodies specific for MUC1-C and MUC1-N (Li et al.,1998). For GST “pull-down” assays, GST or GST-galectin-3 was incubatedwith cell lysates for 2 hours at 4° C. and precipitated withglutathione-Sepharose™ 4B. Precipitates were subjected to immunoblottingwith anti-MUC1-C, anti-galectin-3 (Santa Cruz Biotechnology, Santa Cruz,Calif.), anti-MUC1-N (DF3) (Ren et al., 2004), anti-EGFR (Santa CruzBiotechnology), or anti-β-actin.

Plasmid Constructions.

The pIRESpuro2-MUC1 and pIRESpuro2-MUC1-C vectors have been described(Huang et al. (2003); Li et al. (2001)). pRNA-U6.1/Neo-MUC1siRNAplasmids were constructed by ligation of MUC1siRNA#2(AAGGTACCATCAATGTCCACG (SEQ ID NO:3), MUC1siRNA#4 (AAGTTCAGTGCCAGCTCTAC(SEQ ID NO:5) or random control (CsiRNA; CGCTTACCGATTCAGAATGG (SEQ IDNO:6) sequences into pRNA-U6.1 (GenScript). pCR3.1-hFc-MUC1-Cextracellular domain (MUC1-C/ED) was constructed by PCR amplification ofMUC1-C/ED from pIRESpuro2-MUC1-C and cloned into the mammalian pCR3.1vector. The human Fc fragment (hFc) was amplified from the CD5-IgG1plasmid (Aruffo et al., 1990). The N36A mutation in pIRESpuro2-MUC1-Cwas introduced using the QuickChange™ kit (Stratagene, La Jolla,Calif.). For vectors encoding galectin-3 and galectin-3 fragments, cDNAswere synthesized from ZR-75-1 mRNA using specific primers and clonedinto the PET22b+vector (Novagen, San Diego, Calif.). The plasmidsexpressing GST-galectin-3 or GST-galectin-3 fragments were generated byPCR from PET22b+-galectin-3 and cloned into the pGEX4T1 vector (AmershamPharmacia Biotech, Piscataway, N.J.). The galectin-3 promoter regionsspanning −3000 to +141 and −836 to +141 bp from the transcription startsite were amplified using the failsafe PCR kit (Epicentre, Madison,Wis.) and cloned into the pGL3 basic vector NheI and HindIII sites. Thegalectin-3 3′-UTR was cloned into the pMIR reporter plasmid (Ambion,Austin, Tex.).

In Vitro Binding Assays.

hFc and hFc-MUC1-C/ED were incubated with purified galectin-3 in lysisbuffer for 1 hour at 4° C. The reaction products were precipitated withprotein G-Sepharose™ and immunoblotted with anti-galectin-3. In otherstudies, hFc-MUC1-C/ED was incubated with GST-galectin-3 deletionmutants. The reaction products were precipitated withglutathione-sepharose 4B and immunoblotted with anti-MUC1-C/ED (Ren etal., 2004).

Cell Transfections.

pIRESpuro2, pIRESpuro2-MUC1, pIRESpuro2-MUC1-C andpIRESpuro2-MUC1-C(N36A) were transfected into BT549 cells with FuGENE™ 6reagent (Roche, Basel, Switzerland) and stable clones were selected inthe presence of 300 ng/ml puromycin (Calbiochem-Novabiochem, La Jolla,Calif.). The pCR3.1-hFc-MUC1-C/ED plasmid was transfected into CHO-K1cells with FuGENE™ 6 reagent and selected in the presence of 1 mg/mlG418 (Invitrogen, Carlsbad, Calif.). ZR-75-1 cells were stablytransfected to express an empty vector of a MUC1 siRNA as described (Renet al., 2004). Transient transfections with a MUC1 siRNA pool,galectin-3 siRNA pool, or a non-specific control duplex IX RNA(Dharmacon, Lafayette, Colo.) were performed in the presence ofLipofectamine™ 2000 (Invitrogen, Carlsbad, Calif.). DU145 cells weretransfected with pRNA-U6.1/Neo-CsiRNA, pRNA-U6.1/Neo-MUC1siRNA#2 orpRNA-U6.1/Neo-MUC1siRNA#4 and selected in 200 μg/ml neomycin. DU145/MUC1siRNA#4 cells were transfected with pIRES-puro2 or pIRES-puro2-MUC1-Cand selected in the presence of 400 ng/ml puromycin.

Quantitative Real-Time PCR.

Total RNA was extracted from cells with TRIzol™ reagent (Invitrogen,Carlsbad, Calif.) and reverse transcribed with oligo(dT) priming andSuperscript™ III reverse transcriptase (Invitrogen, Carlsbad, Calif.).Quantification of galectin-3 transcripts was performed with primer pairsCAACCAGTACTTGTATTTTGAATG (SEQ ID NO:16) and CAATGAGAACAACAGGAGAGTCA (SEQID NO:17) using Power SYBk Green QPCR Mastermix™ and ABI Prism™ 7000sequence detections system (Applied Biosystems. Foster City, Calif.).Relative gene expression was determined using the comparative ddCT(threshold) method with GAPDH as the internal control.

Nuclear Run-on Assays.

Nuclei were isolated from 5×10⁶ cells and assayed as described (Patroneet al., 2000). In brief, run-on transcription was performed in thepresence of biotin-16-UTP (Roche Applied Science) for 30 min at 30° C.RNA was purified using TRIzol reagent. The biotinylated RNA was isolatedwith Dynabeads M-280 (Invitrogen). Galectin-3 and, as an endogenouscontrol, β-actin nascent transcripts were analyzed by quantitativeRT-PCR. The difference in transcript level was calculated by thecomparative C_(T) method.

mRNA Stability Assays.

Cells were treated with 1.3 μg/ml actinomycin D and harvested atdifferent intervals. Total RNA was isolated and analyzed for galectin-3and GAPDH mRNA levels by quantitative RT-PCT. The half life ofgalectin-3 mRNA was calculated using linear regression analysis.

Luciferase Assays.

Cells were transfected with pGL3-galectin-3 promoter constructspGal-3(−3000/+141)-Luc or pGal-3(−836/+141)-Luc and pcDNA-LacZ usingFuGENE™ 6 reagent (Roche Diagnostics, Indianapolis, Ind.). Galectin-3promoter activity was measured 24 hours after transfection. To assessfunction of the galectin-3 3′UTR, cells were transfected withpMIR-Gal-3(3′UTR) and pMIR-β-galactosidase using FuGENE™ 6 reagent andassayed at 48 hours after transfection. Luciferase assays were performedwith the luciferase assay system (Promega Corp., Madison, Wis.). Theresults were normalized to β-galactosidase activity and presented asrelative luciferase activity.

Analysis of miR-322 Expression.

Northern blotting for detection of miRNAs was performed as described(Lee and Ambros, 2001; Johnson et al., 2005). Total cellular RNA (40 μg)isolated using TRIzol™ reagent (Invitrogen, Carlsbad, Calif.) was sizefractionated in 15% urea-acrylamide gels. The RNA was transferred topositively charged nylon membranes (BrightStar™-Plus, Ambion, Austin,Tex.) by electroblotting. After UV-crosslinking and prehybridization,the RNA was hybridized to ³²P-labeled probes against miR-322(GCAGCGCCTCACGTTT) (SEQ ID NO:18) and U6-snRNA (GCGTGTCATCCTTGCGCAG)(SEQ ID NO:19) (Starfire™ Labeling System; Integrated DNA Technologies,Coralville, Iowa).

Antisense inhibition of miR-322. An antisense 2′-O-methyloligoribonucleotide (GGUAUAGCAGCGCCUCACGUUUUG) (SEQ ID NO:20) againstmiR-322 and a scrambled 2′-O-methyl oligoribonucleotide(CACGGAUCUAGGUCGUAACGUAGG) (SEQ ID NO:21) (Integrated DNA Technologies)were transfected into BT549 cells (50 pmol/10,000 cells) in the presenceof Lipofectamine™ 2000. At 72 hours after transfection, the cells wereanalyzed for galectin-3 expression.

Protein Purification.

CHO-K1 cells expressing hFc-MUC1-C/ED were adapted to grow as asuspension culture in mAb production medium (BD Biosciences, San Jose,Calif.) using the Cell Line 1000 chamber flask (Nunc, Rochester, N.Y.).The secreted hFc-MUC1-C/ED protein was purified using a protein-A column(Pierce Biotechnology, Rockford, Ill.). The galectin-3 proteins werepurified from E. coli using an Asialofetulin column as described(Pelletier and Sato, 2002).

Identification of MUC1-C/ED Cell Surface Binding Proteins.

Purified hFc-MUC1-C/ED and hFc (US Biologicals) were cross-linked toagarose beads. ZR-75-1 cells were grown in 0.1% FBS for 48 hours andwashed with salt solution (10 mM MES, pH 6.2, 750 mM NaCl, 2 mM EDTA).The salt extract was passed through a 0.45 μm filter, diluted 3× inwater, adjusted to 1 mM CaCl₂/3 mM MgCl2 (pH 7.4) and passed through atandem hFc-MUC1-C/ED and control hFc column. The column was washed with10 mM Tris-HCl, pH 7.4, 250 mM NaCl, 1 mM CaCl₂ and 3 mM MgCl₂, andeluted with 2 M NaCl and 5 mM EDTA. The eluate was dialyzed against PBS,concentrated, and analyzed by SDS-PAGE and silver staining.

Surface plasmon resonance analysis. Surface plasmon resonancemeasurements were determined using a BIACORE™ 3000 instrument at 25° C.Galectin-3 and galectin-3(63-250) were covalently coupled to CM5Biacore™ sensor chips (500-1000 RU) at 30 μg/ml using the Biacore™ AmineCoupling kit. hFc-MUC1-C/ED and hFc diluted in running buffer (50 mMTris-HCl, pH 7.4, 150 mM NaCl, 0.005% NP-40) were injected acrossgalectin-3, galectin-3(63-250) or control flow cells at a rate of 30μl/min. Regeneration of the sensor surface was performed with a30-second pulse of 100 mM lactose. Association and dissociation kineticconstants were calculated by BIAevaluation™ software using 1:1 bindingwith a drifting base model.

In Vitro Deglycosylation.

MUC1-C was immunoprecipitated from cell lysates, eluted from the beadswith 50 mM glycine-HCl, pH 2.6, dialyzed against 50 mM sodium phosphatebuffer, pH 7.0, and deglycosylated under denaturing conditions using theGlycokit™ glycosylation analysis kit (ProZyme, San Leandro, Calif.).

Immunofluorescence Microscopy.

Cells were fixed in 3.7% formaldehyde, permeabilized in 0.2% Triton™X-100 and post-fixed in 3.7% formaldehyde. The cells were blocked with10% normal goat serum and stained with antibodies specific for MUC1-N(MAb DF3) and antibodies specific for EGFR (Santa Cruz Biotechnology,Santa Cruz, Calif.), followed by fluorescein-conjugated antibodiesspecific for rabbit IgG or Texas Red-conjugated antibodies specific formouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.). Thecells were then mounted onto coverslips using slow-fade mounting reagent(Molecular Probes, Carlsbad, Calif.). Images were captured with a ZeissLSM510 confocal microscope under 63× magnification and 1024×1024resolution.

Ectopic Expression of Pre-mIR-322.

Pre-miR constructs encoding mature miR-322 (AAACGUGAGGCGCUGCUAUA; SEQ IDNO:7) or a scrambled control sequence (GUCAGGUCAAACGGCCUAGAU; SEQ IDNO:8) were cloned into pcDNA 6.2-GW/miR vector (BLOCK-iT Pol II miR RNAiExpression Vector Kit; Invitrogen). The constructed clones wereintroduced into cells using Fugene6. The transfected cells were selectedin 10 μg/ml blasticidin.

Example 2

MUC1 Upregulates Galectin-3 Expression in Carcinoma Cells MUC1 andgalectin-3 are both widely expressed in human carcinomas. To assesscoexpression of MUC1 and galectin-3, lysates from diverse humancarcinoma cells were analyzed. Cells expressing MUC1 were also positivefor galectin-3 expression (FIG. 1A). The relationship between MUC1 andgalectin-3 expression was further assessed in BT549 breast cancer cells,which are null for MUC1 and have low levels of galectin-3 (FIG. 1B).Stable expression of MUC1, but not the empty vector, was associated withsubstantial upregulation of galectin-3 (FIG. 1B). MUC1-dependentregulation of galectin-3 expression was also assessed in ZR-75-1 breastcancer cells that express endogenous MUC1 (Ren et al., 2004). Notably,stable silencing of MUC1 with a MUC1 siRNA (Ren et al., 2004) wasassociated with decreases in galectin-3 expression (FIG. 1C). Transientsilencing of MUC1 with a pool of MUC1 siRNAs also resulted indownregulation of galectin-3 expression (FIG. 1D). Moreover, stablesilencing of MUC1 in DU145 prostate cancer cells was associated withdownregulation of galectin-3 (FIG. 1D). To exclude off-target effects ofthe MUC1siRNA#2, which is directed against sequences encoding MUC1-C, weused MUC1siRNA#4. Silencing MUC1 with MUC1siRNA#4 similarly resulted indownregulation of galectin-3 expression (FIG. 1E). MUC1siRNA#4 targetssequences in the MUC1-N coding region. Consequently, we transfectedthese cells to express MUC1-C (FIG. 1E). Notably, rescue of MUC1-Cexpression was associated with increases in galectin-3 (FIG. 1E). Thesefindings indicate that MUC1-C upregulates galectin-3 expression.

These findings indicate that MUC1 upregulates galectin-3 expression.

Example 3 MUC1 stabilizes galectin-3 transcripts

To define how MUC1 regulates galectin-3 expression, semi-quantitativeRT-PCR analysis of galectin-3 mRNA levels was performed. BT549 cellsstably expressing MUC1 exhibited increases in galectin-3 transcripts ascompared to that in BT549/vector cells (FIG. 2A, left). MUC1-dependentupregulation of galectin-3 mRNA levels in BT549 cells was confirmed byquantitative RT-PCR (FIG. 2A, right). In concert with these results,stable silencing of MUC1 in ZR-75-1 cells decreased galectin-3 mRNAlevels as determined by semi-quantative (FIG. 2B, left) and quantitative(FIG. 2B, right) RT-PCR. Stable silencing of MUC1 in DU145 cells alsodecreased galectin-3 mRNA levels (FIG. 2C, left). Moreover, expressionof MUC1-C in DU145/MUC1siRNA#4 cells was associated with increases ingalectin-3 transcripts (FIG. 2C, right). To determine whether MUC1activates galectin-3 gene transcription, regions (−3000 to +141 and −836to +141) of the galectin-3 promoter were ligated upstream to aluciferase (Luc) reporter. The results obtained from transfectingpGal-3(−3000/+141)-Luc into BT549/vector and BT549/MUC1 cells indicatedthat MUC1 has little if any effect on activation of the galectin-3promoter (FIG. 2F). MUC1 also had no apparent effect on activation ofthe pGal-3(−3000/+141)-Luc or pGal-3(−836/+141)-Luc constructs inZR-75-1 cells (FIGS. 2G and 2H), indicating that MUC1 upregulatesgalectin-3 mRNA levels by a post-transcriptional mechanism. To extendthis analysis to the endogenous galectin-3 gene, nuclear run-on assayswere performed. The rate of galectin-3 gene transciption in BT549 cellswas unaffected by stable expression of MUC1 (FIG. 21, left). Inaddition, silencing of MUC1 in ZR-75-1 cells had little if any effect ontranscription of the galectin-3 gene (FIG. 21, right). These findingsindicate that MUC1 increases galectin-3 mRNA levels by aposttranscriptional mechanism. To define the mechanism by which MUC1increases galectin-3 mRNA levels, the stability of galectin-3transcripts in BT549/vector and BT549/MUC1 cells was analyzed.Galectin-3 and GAPDH mRNA levels were assayed by quantitative RT-PCRafter inhibiting transcription with actinomycin D. The rate ofgalectin-3 mRNA degradation in BT549/vector cells was increased ascompared to that in BT549/MUC1 cells, indicating that MUC1 stabilizesgalectin-3 transcripts (FIG. 2D). In this regard, the half-lives ofgalectin-3 transcripts were 22.8 and 12.0 h in the presence and absenceof MUC1, respectively. To determine if the galectin-3 mRNA 3′untranslated region (3′UTR) is regulated by MUC1, the 3′UTR was ligateddownstream to the luciferase gene in the pMIR reporter plasmid (FIG.2E). Transfection of BT549 cells with pMIR-Gal-3(3′UTR) demonstratedthat MUC1 increases expression of the luciferase reporter (FIG. 2E,left). Expression of pMIR-Gal-3(3′UTR) was also increased in ZR-75-1cells by a MUC1-dependent mechanism (FIG. 2E, right). These findingsindicate that MUC1 stabilizes galectin-3 transcripts by a mechanisminvolving the 3′UTR.

Example 4 MUC1 Suppresses miR-322 Expression and Thereby IncreasesStability of Galectin-3 mRNA

The galectin-3 3′UTR has no identifiable AU-rich elements that functionin the regulation of mRNA stability. Consequently, studies wereconducted to determine if the galectin-3 3′UTR contains sequences thatcould be targeted by a microRNA. A search of the Sanger miRNA registry(http://microrna.sanger.ac.uk) identified miR-322 as a possiblecandidate. miR-322 is expressed from a stem-loop structure (pre-mIR-322)at which miR-424 also originates (FIG. 3A). Expression of miR-424 hasbeen demonstrated in human cells (Kasashima et al., 2004); however,there is no available evidence for a human miR-322. Alignment of mouse,rat and the putative human miR-322 revealed conservation of thesequences (FIG. 3B). In addition, human miR-322 has recognitionsequences for the human galectin-3 3′UTR (FIG. 3B). Northern blotanalysis of RNA from BT549/vector cells demonstrated expression ofmiR-322 (FIG. 3C, left). By contrast, it was found that miR-322 levelsare decreased in BT549 cells that express MUC1 (FIG. 3C, left). Studieswith ZR-75-1 cells further showed that miR-322 expression is suppressedby a MUC1-dependent mechanism (FIG. 3C, middle). Moreover, silencingMUC1 in DU145 cells with MUC1siRNA#2 or MUC1siRNA#4 increased miR-322expression (FIG. 3C, right). These results thus demonstrate that MUC1suppresses miR-322 expression. To determine whether miR-322 regulatesgalectin-3 expression, BT549/vector cells were transfected with anantisense 2′-O-methyl oligoribonucleotide targeted against miR-322 or,as a control, with a scrambled 2′-β-methyl oligoribonucleotide. It wasfound that anti-miR-322, and not the scrambled oligo, upregulatesgalectin-3 expression at the mRNA (FIG. 3F, left) and protein levels(FIG. 3F, right). Stability of galectin-3 transcripts was alsodetermined by quantitative RT-PCR after actinomycin D treatment.Degradation of galectin-3 mRNA was increased in cells transfected withthe scrambled oligo compared to that obtained with anti-miR-322 (FIG.3D), indicating that miR-322 decreases galectin-3 mRNA stability. Thehalf-lives of galectin-3 transcripts were 13.0 and 19.5 h in cellstransfected with the scrambled oligo and anti-miR-322, respectively. Asanother approach, BT549/MUC1 cells were transfected with a pre-mIR-322or a scrambled miRNA. Ectopic miR-322 had no detectable effect ongalectin-3 gene transcription as determined by run-on assays (FIG. 3G).Moreover, analysis of galectin-3 expression demonstrated that theectopic miR-322 decreases galectin-3 mRNA and protein levels (FIG. 3E).These findings indicate that miR-322 decreases stability of galectin-3transcripts.

Example 5 Direct Binding of the MUC1-C Extracellular Domain andGalectin-3

Galectin-3 binds to glycans on cell surface molecules like MUC1. Todetermine if the upregulation of galectin-3 expression is associatedwith binding of MUC1 and galectin-3, lysates from ZR-75-1 cells wereimmunoprecipitated with anti-MUC1-C or, as a control, IgG. Galectin-3was detectable in the anti-MUC1-C, and not the IgG, precipitates (FIG.4A, left). Incubation of a GST-galectin-3 fusion protein with ZR-75-1cell lysates in pull-down experiments further demonstrated binding ofMUC1-C and galectin-3 (FIG. 4A, right). MUC1-C consists of a 58 aminoacid extracellular domain (ED), a 28 amino acid transmembrane domain(TM) and a 72 amino acid cytoplasmic domain (CD) (FIG. 4B). The MUC1-Cextracellular domain (MUC1-C/ED) contains three potentialN-glycosylation sites that could confer the interaction between MUC1-Cand galectin-3 (FIG. 4B). To determine if galectin-3 associates with theMUC1-C/ED, the MUC1-C/ED was fused to human Fc (hFc-MUC1-C/ED) (FIG.4B). hFc and hFc-MUC1-C/ED bound to agarose beads were incubated withsupernatants from ZR-75-1 breast cancer cells. Analysis of adsorbedproteins by SDS-PAGE and silver staining demonstrated binding of a ˜26kDa protein to hFc-MUC1-C/ED and not hFc (FIG. 4C, left). Digestion ofthe adsorbed protein with trypsin and mass spectroscopy analysis of thetryptic peptides supported identity with galectin-J. Immunoblot analysisof the adsorbed protein also confirmed the association of MUC1-C/ED andgalectin-3. To determine if MUC1-C/ED and galectin-3 interact directly,hFc or hFc-MUC1-C/ED were incubated with purified recombinantgalectin-3. Analysis of the adsorbates by immunoblotting withanti-galectin-3 demonstrated binding of galectin-3 to hFc-MUC1-C/ED andnot hFc (FIG. 4C, right). To define the region of galectin-3 thatconfers binding to MUC1-C/ED, GST fusion proteins with full-lengthgalectin-3 or certain deletion mutants were prepared (FIG. 4D).MUC1-C/ED was pulled-down with GST-galectin-3 and GST-galectin-3(63-250)that contains the CRD (FIG. 4D). By contrast, there was no detectablebinding of MUC1-C/ED and GST-galectin-3(1-62) or GST-galectin-3(1-138)that include the ND (FIG. 4D). The kinetics of the interaction betweenMUC1-C/ED and galectin-3 were assessed by immobilizing galectin-3 to asensor chip and assaying for binding of MUC1-C/ED in a BIAcore (FIG.4E). MUC1-C/ED bound to galectin-3 with a dissociation constant (KD) of11.1 nM. Similar kinetics (KD=9.8 nM) were obtained for binding ofMUC1-C/ED to galectin-3(63-250) (FIG. 4F). These findings indicate thatMUC1-C/ED and galectin-3 bind directly and that this interaction ismediated by the galectin-3 CRD.

Example 6 MUC1-C Glycosylation Is Necessary for the Galectin-3Interaction

Galectin-3 binds to both β-galactoside-containing glycoproteins andnon-glycosylated proteins (Shimura et al., 2004). MUC1-C is expressed as25-20, 17 and 15 kDa species (FIG. 5A); however, it is not known if anyof these species are subject to glycosylation. To assess MUC1-Cglycosylation, anti-MUC1-C precipitates from BT549/MUC1 cells wereincubated in the absence and presence of N-glycosidases. Deglycosylationwas associated with a decrease in the broad anti-MUC1-C band at 25-20kDa and an increase in the 17 kDa band (FIG. 5A, left), consistent withthe presence of N-linked glycans. Similar results were obtained whenendogenous MUC1-C from ZR-75-1 cells was incubated with N-glycosidases(FIG. 5A, right). To extend this analysis, MUC1-C was expressed inwild-type and glycosylation-deficient CHO cells. Expression of MUC1-C inCHO-Lec1 cells, which are deficient in N-glycosylation, was associatedwith loss of the 25-20 kDa band (FIG. 5B, left). A similar pattern ofMUC1-C expression was observed in CHO-Lec8 cells, which are deficientfor incorporation of β-galactosides (FIG. 5B, left). Notably,GST-galectin-3 pull-downs showed binding only to the 25-20 kDa MUC1-Cexpressed in CHO-K1 cells (FIG. 5B, right), consistent with interactionof galectin-3 and N-linked glycans on MUC1-C. The MUC1-C extracellulardomain contains three asparagine residues (positions 16, 25 and 36; seeFIG. 4B), one of which (NLT) conforms to a predicted N-glycosylationsite. Consequently, wild-type MUC1-C or MUC1-C with a N36A mutation wasstably expressed in human BT549 cells. Expression of wild-type MUC1-Cwas associated with anti-MUC1-C reactivity that was predominant at 25-20and 15 kDa (FIG. 5C left). By contrast, expression of MUC1-C(N36A)resulted in anti-MUC1-C reactivity at 17 and 15 kDa (FIG. 5C, left),consistent with loss of the glycosylated 25-20 kDa species. As acontrol, deglycosylation of wild-type MUC1-C was also associated withreactivity at 17 and 15 kDa (FIG. 5C, right). Importantly, binding ofgalectin-3 was detectable with wild-type MUC1-C, but not withMUC1-C(N36A) (FIG. 5D). These findings indicate that galectin-3 binds toMUC1-C glycosylated at the Asn-36 site.

Example 7 MUC1-C Subunit Induces Galectin-3 Expression

To further define the interaction between MUC1-C and galectin-3, BT549cells stably transfected with MUC1-C or MUC1-C(N36A) were analyzed forinduction of galectin-3 (FIG. 6A). The results demonstrate that MUC1-Cis sufficient to upregulate galectin-3 expression (FIG. 6A). Bycontrast, MUC1-C(N36A) had little effect on galectin-3 levels (FIG. 6A).Semi-quantitative and quantitative RT-PCR also demonstrated thatMUC1-C-induced upregulation of galectin-3 mRNA levels is substantiallyattenuated with MUC1-C(N36A) (FIG. 6B). As found with MUC1, there was noapparent effect of MUC1-C or MUC1-C(N36A) on activation of thegalectin-3 promoter. However, MUC1-C, but not MUC1-C(N36A), waseffective in increasing expression of the pMIR-Gal-3(3′UTR) reporter(FIG. 6C). In concert with these results, it was found thatMUC1-C-induced suppression of miR-322 is also abrogated by the N36Amutation (FIG. 6D). These findings indicate that glycosylation of MUC1-CAsn-36 is necessary for suppression of miR-322 expression andupregulation of galectin-3.

Example 8 Galectin-3 Confers the Interaction Between MUC1 and EpidermalGrowth Factor Receptor (EGFR)

To determine if galectin-3 functions in the interaction between MUC1 andEGFR, ZR-75-1 cells were treated with lactose, a competitive inhibitorof galectin-3 binding and, as a control, with sucrose. EGF-stimulatedbinding of MUC1 and EGFR was attenuated by lactose and not sucrose,indicating that galectin-3 may facilitate this interaction (FIG. 7A). Toextend the analysis, the ZR-75-1 cells were transfected with a pool ofgalectin-3 specific siRNAs or a control siRNA. The results demonstratethat silencing galectin-3 blocks the EGF-induced association of MUC1 andEGFR (FIG. 7B). To relate these studies to the downregulation ofgalectin-3 by miR-322, ZR-75-1 cells were transfected with pre-mIR-322or a scrambled miRNA. As found in BT549/MUC1 cells (FIG. 3E), ectopicmiR-322 decreased galectin-3 expression (FIG. 7C). Moreover,downregulation of galectin-3 by ectopic miR-322 attenuated theEGF-induced interaction between MUC1 and EGFR (FIG. 7D). EGF stimulationis associated with increased colocalization of MUC1 and EGFR at the cellmembrane (Li et al., 2001b). As shown previously by confocal microscopy(Li et al., 2001b), EGFR and MUC1 are uniformly distributed over thecell membrane of control ZR-75-1 cells (Supplemental FIG. 5E). FollowingEGF stimulation, EGFR and MUC1 cluster in patches (Li et al., 2001b).The colocalization of EGFR and MUC1 in clusters was blocked by lactoseand not sucrose (FIG. 5E). Consistent with involvement of galectin-3,EGF-induced clustering of EGFR and MUC1 was also blocked by silencinggalectin-3 (FIG. 5F). These findings indicate that MUC1 suppression ofmiR-322 and thereby upregulation of galectin-3 is of importance to theinteraction between MUC1 and EGFR.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of some embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-14. (canceled)
 15. A method of inhibiting the association of MUC1 withepidermal growth factor receptor (EGFR) in a cancer cell that expressesMUC1, the method comprising: contacting the cancer cell with a compoundthat inhibits binding of galectin-3 to the extracellular domain ofMUC1-C.
 16. The method of claim 15, wherein the compound comprises apeptide fragment of MUC1-C.
 17. The method of claim 15, wherein thecompound comprises a peptide fragment of the extracellular domain ofMUC1-C.
 18. The method of claim 17, wherein the compound is apolypeptide comprising a peptide fragment of the extracellular domain ofMUC1-C fused to an immunoglobulin Fc region. 19-20. (canceled)
 21. Themethod of claim 15, wherein the compound is an antibody, or an antibodyfragment, that binds to or the extracellular domain of MUC1-C.
 22. Amethod of inhibiting activation of galectin-3 expression in a cancercell that expresses MUC1, the method comprising: contacting the cancercell with a compound that inhibits activation of galectin-3 expressionby MUC1.
 23. The method of claim 22, wherein the compound comprises anucleic acid having the sequence of miR-322. 24-31. (canceled)
 32. Themethod of claim 31, wherein the small molecule comprises a nucleic acidaptamer.
 33. The method of claim 31, wherein the small molecule consistsof a nucleic acid aptamer. 34-44. (canceled)
 45. A method of inhibitingexpression of galectin-3 in a cancer cell that expresses MUC1, themethod comprising: (a) identifying a subject as having a cancercomprising a cancer cell that expresses MUC1; and (b) introducing intothe cell a nucleic acid that inhibits the expression of MUC1.
 46. Themethod of claim 45, wherein the nucleic acid is an antisense nucleicacid, a small interfering RNA (siRNA), or a nucleic acid that directsthe expression of an antisense nucleic acid or siRNA.
 47. The method ofclaim 45, wherein the introducing step comprises administration of thenucleic acid to the cancer cell and uptake of the nucleic acid by thecancer cell.
 48. The method of claim 45, wherein the introducing stepcomprises administering to a subject mammalian subject, and uptake bythe cancer cell of, a nucleic acid: (i) from which sense and anti-sensestrands of the siRNA can be transcribed under the direction of separateIREs; or (ii) from which both sense and anti-sense strands of the siRNAcan be transcribed under the direction of a single TRE.
 49. The methodof claim 45, wherein the subject is a human patient.
 50. The method ofclaim 45, wherein the cancer cell is a breast cancer cell. 51.(canceled)
 52. A method of promoting apoptosis of a cell, the methodcomprising: determining whether the cell expresses MUC1; and if the cellexpresses MUC1, contacting the cell with a compound that inhibitsphosphorylation of galectin-3 by casein kinase
 1. 53. The method ofclaim 52, wherein the cell is a cancer cell.